Microorganisms and methods for the biosynthesis of adipate, hexamethylenediamine and 6-aminocaproic acid

ABSTRACT

The invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway. The invention additionally provides a method for producing 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid. The method can include culturing a 6-aminocaproic acid, caprolactam or hexametheylenediamine producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid pathway enzyme in a sufficient amount to produce the respective product, under conditions and for a sufficient period of time to produce 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid.

This application is a divisional of U.S. application Ser. No.15/263,149, filed Sep. 12, 2016, which is a continuation of U.S.application Ser. No. 13/730,612, filed Dec. 28, 2012, now issued U.S.Pat. No. 9,458,480, which is a continuation of U.S. application Ser. No.12/776,365, filed May 7, 2010, now issued U.S. Pat. No. 8,377,680, whichclaims the benefit of priority of U.S. Provisional Application Ser. No.61/176,196, filed May 7, 2009, U.S. Provisional Application Ser. No.61/219,365, filed Jun. 22, 2009, U.S. Provisional Application Ser. No.61/244,844, filed Sep. 22, 2009, U.S. Provisional Application Ser. No.61/246,973, filed Sep. 29, 2009, and U.S. Provisional Application Ser.No. 61/247,533, filed Sep. 30, 2009, each of which the entire contentsof are incorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Dec. 7, 2018, is named12956-469-999_SeqList.txt and is 33,776 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having adipate, hexamethylenediamine,6-aminocaproic acid and caprolactam biosynthetic capability.

Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14. Itcan be used is to produce nylon 6,6, a linear polyamide made bycondensing adipic acid with hexamethylenediamine. This is employed formanufacturing different kinds of fibers. Other uses of adipic acidinclude its use in plasticizers, unsaturated polyesters, and polyesterpolyols. Additional uses include for production of polyurethane,lubricant components, and as a food ingredient as a flavorant andgelling aid.

Historically, adipic acid was prepared from various fats usingoxidation. Some current processes for adipic acid synthesis rely on theoxidation of KA oil, a mixture of cyclohexanone, the ketone or Kcomponent, and cyclohexanol, the alcohol or A component, or of purecyclohexanol using an excess of strong nitric acid. There are severalvariations of this theme which differ in the routes for production of KAor cyclohexanol. For example, phenol is an alternative raw material inKA oil production, and the process for the synthesis of adipic acid fromphenol has been described. The other versions of this process tend touse oxidizing agents other than nitric acid, such as hydrogen peroxide,air or oxygen.

In addition to hexamethylenediamine (HMDA) being used in the productionof nylon-6,6 as described above, it is also utilized to makehexamethylene diisocyanate, a monomer feedstock used in the productionof polyurethane. The diamine also serves as a cross-linking agent inepoxy resins. HMDA is presently produced by the hydrogenation ofadiponitrile.

Caprolactam is an organic compound which is a lactam of 6-aminohexanoicacid (ε-aminohexanoic acid, 6-aminocaproic acid). It can alternativelybe considered cyclic amide of caproic acid. One use of caprolactam is asa monomer in the production of nylon-6. Caprolactam can be synthesizedfrom cyclohexanone via an oximation process using hydroxylammoniumsulfate followed by catalytic rearrangement using the Beckmannrearrangement process step.

Methods for effectively producing commercial quantities of compoundssuch as hexamethylenediamine, 6-aminocaproic acid, levulinic acid andcarpolactamare described herein and include related advantages.

SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organismhaving a 6-aminocaproic acid, caprolactam or hexametheylenediaminepathway. The microbial organism contains at least one exogenous nucleicacid encoding an enzyme in the respective 6-aminocaproic acid,caprolactam, hexametheylenediamine or levulinic acid pathway. Theinvention additionally provides a method for producing 6-aminocaproicacid, caprolactam or hexametheylenediamine. The method can includeculturing a 6-aminocaproic acid, caprolactam, hexametheylenediamine orlevulinic acid producing microbial organism, where the microbialorganism expresses at least one exogenous nucleic acid encoding a6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinicacid pathway enzyme in a sufficient amount to produce the respectiveproduct, under conditions and for a sufficient period of time to produce6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinicacid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary pathway for adipate degradation in theperoxisome of Penicillium chrysogenum.

FIG. 2 shows an exemplary pathway for adipate formation via a reversedegradation pathway. Several options are provided for the finalconversion of adipyl-CoA to adipate.

FIG. 3 shows an exemplary pathway for adipate formation via the3-oxoadipate pathway.

FIG. 4 show the similar enzyme chemistries of the last three steps ofthe 3-oxoadipate pathway for adipate synthesis and the reductive TCAcycle.

FIG. 5 shows an exemplary pathway for synthesis of adipic acid fromglucose via cis,cis-muconic acid. Biosynthetic intermediates(abbreviations): D-erythrose 4-phosphate (E4P), phosphoenolpyruvic acid(PEP), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP),3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), protocatechuicacid (PCA). Enzymes (encoding genes) or reaction conditions: (a) DAHPsynthase (aroFFBR), (b) 3-dehydroquinate synthase (aroB), (c)3-dehydroquinate dehydratase (aroD), (d) DHS dehydratase (aroZ), (e)protocatechuate decarboxylase (aroY), (f) catechol 1,2-dioxygenase(catA), (g) 10% Pt/C, H2, 3400 kPa, 25° C. Figure taken from Niu et al.,Biotechnol. Prog. 18:201-211 (2002)).

FIG. 6 shows an exemplary pathway for adipate synthesis viaalpha-ketoadipate using alpha-ketoglutarate as a starting point.

FIG. 7 shows an exemplary pathway for synthesis of adipate using lysineas a starting point.

FIG. 8 shows an exemplary caprolactam synthesis pathway using adipyl-CoAas a starting point.

FIG. 9 shows exemplary adipate synthesis pathways usingalpha-ketoadipate as a starting point.

FIG. 10 shows exemplary pathways from succinyl-CoA and acetyl-CoA tohexamethylenediamine (HMDA) and caprolactam. Pathways for the productionof adipate, 6-aminocaproate, caprolactam, and hexamethylenediamine fromsuccinyl-CoA and acetyl-CoA are depicted. Abbreviations: A)3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C)3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase,E) 3-oxoadipyl-CoA/acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G)3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I)3-hydroxyadipate dehydratase, J) 5-carboxy-2-pentenoate reductase, K)adipyl-CoA/acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoAhydrolase, N) adipyl-CoA reductase (aldehyde forming), 0)6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q)6-aminocaproyl-CoA/acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase,S) amidohydrolase, T) spontaneous cyclization, U) 6-aminocaproyl-CoAreductase (aldehyde forming), V) HMDA transaminase, W) HMDAdehydrogenase.

FIG. 11 shows exemplary pathways from 4-aminobutyryl-CoA and acetyl-CoAto hexamethylenediamine and caprolactam. Pathways for the production of6-aminocaproate, caprolactam, and hexamethylenediamine from4-aminobutyryl-CoA and acetyl-CoA are depicted. Abbreviations: A)3-oxo-6-aminohexanoyl-CoA thiolase, B) 3-oxo-6-aminohexanoyl-CoAreductase, C) 3-hydroxy-6-aminohexanoyl-CoA dehydratase, D)6-aminohex-2-enoyl-CoA reductase, E) 3-oxo-6-aminohexanoyl-CoA/acyl-CoAtransferase, F) 3-oxo-6-aminohexanoyl-CoA synthase, G)3-oxo-6-aminohexanoyl-CoA hydrolase, H) 3-oxo-6-aminohexanoatereductase, I) 3-hydroxy-6-aminohexanoate dehydratase, J)6-aminohex-2-enoate reductase, K) 6-aminocaproyl-CoA/acyl-CoAtransferase, L) 6-aminocaproyl-CoA synthase, M) 6-aminocaproyl-CoAhydrolase, N) 6-aminocaproyl-CoA reductase (aldehyde forming), 0) HMDAtransaminase, P) HMDA dehydrogenase, Q) spontaneous cyclization, R)amidohydrolase.

FIG. 12 shows pathways to 6-aminocaproate from pyruvate and succinicsemialdehyde. Enzymes are A) HODH aldolase, B) OHED hydratase, C) OHEDreductase, D) 2-OHD decarboxylase, E) adipate semialdehydeaminotransferase and/or adipate semialdehyde oxidoreductase (aminating),F) OHED decarboxylase, G) 6-OHE reductase, H) 2-OHD aminotransferaseand/or 2-OHD oxidoreductase (aminating), I) 2-AHD decarboxylase, J) OHEDaminotransferase and/or OHED oxidoreductase (aminating), K) 2-AHEreductase, L) HODH formate-lyase and/or HODH dehydrogenase, M)3-hydroxyadipyl-CoA dehydratase, N) 2,3-dehydroadipyl-CoA reductase, 0)adipyl-CoA dehydrogenase, P) OHED formate-lyase and/or OHEDdehydrogenase, Q) 2-OHD formate-lyase and/or 2-OHD dehydrogenase.Abbreviations are: HODH=4-hydroxy-2-oxoheptane-1,7-dioate,OHED=2-oxohept-4-ene-1,7-dioate, 2-OHD=2-oxoheptane-1,7-dioate,2-AHE=2-aminohept-4-ene-1,7-dioate, 2-AHD=2-aminoheptane-1,7-dioate, and6-OHE=6-oxohex-4-enoate.

FIG. 13 shows pathways to hexamethylenediamine from 6-aminocapropate.Enzymes are A) 6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C)6-aminocaproic semialdehyde aminotransferase and/or 6-aminocaproicsemialdehyde oxidoreductase (aminating), D) 6-aminocaproateN-acetyltransferase, E) 6-acetamidohexanoate kinase, F) 6-AAHOPoxidoreductase, G) 6-acetamidohexanal aminotransferase and/or6-acetamidohexanal oxidoreductase (aminating), H) 6-acetamidohexanamineN-acetyltransferase and/or 6-acetamidohexanamine hydrolase (amide), I)6-acetamidohexanoate CoA transferase and/or 6-acetamidohexanoate CoAligase, J) 6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOPacyltransferase, L) 6-AHOP acyltransferase, M) 6-aminocaproate CoAtransferase and/or 6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoAoxidoreductase. Abbreviations are:6-AAHOP=[(6-acetamidohexanoyl)oxy]phosphonate and6-AHOP=[(6-aminohexanoyl)oxy]phosphonate.

FIG. 14 shows: A) the acetyl-CoA cycle of arginine biosynthesis.Reactions (1) and (2) are catalyzed by ornithine acetyltransferase withacetylglutamate synthase and ornithine acyltransferase functionality.Reaction 3 is a lumped reaction catalyzed by acetylglutamate kinase,N-acetylglutamylphosphate reductase, and acetylornithineaminotransferase; B) the acetyl-CoA cycle of HMDA biosynthesis.Reactions (1) and (2) are catalyzed by HMDA acetyltransferase. Reaction(3) is a lumped reaction that includes all pathways to6-acetamidohexanamine from 6-acetamidohexanoate shown in FIG. 13.

FIG. 15 shows the growth of E. coli in media containing variousconcentrations of 6-ACA. E. coli was inoculated into media and grown ineither aerobic (left and right bars) or anaerobic (middle bars)conditions. The cultures were grown for 48 hrs during the first trialand 30 hrs for a second trial under aerobic conditions (right bars).

FIG. 16 shows the tolerance of E. coli when exposed to 6-ACA. Midlog(OD600=0.3, lower dashed line) or early stationary (OD600=0.6, upperdashed line) cells were spun down and resuspended in fresh M9-Glucosemedium with various concentrations of 6-ACA. After overnight growth,cultures were measured for growth by measuring OD600.

FIG. 17 shows the ethanol production from cultures exposed to variousconcentrations of 6-ACA. Midlog or early stationary cells were spun downand resuspended in fresh M9-Glucose medium with various concentrationsof 6-ACA. After overnight growth, cultures were measured for growth bymeasuring OD600 and metabolic activity assayed by ethanol production.

FIG. 18, panels A and B, show the growth in various concentrations of6-ACA with and without glycine betaine. Panel A. OD600 measurements ofmedium inoculated with midlog cultures of E. coli with variousconcentrations of 6-ACA with (right bars) and without (left bars) 2 mMglycine betaine. Panel B. Photograph showing the growth of same culturesin the anaerobic bottles.

FIG. 19 shows LC/MS analysis of in vitro thiolase reactions.Succinyl-CoA and acetyl-CoA were added to His-tagged, purified thiolasesat a ratio of 2:1 (succinyl-CoA:acetyl-CoA). Reactions were analyzed byLC/MS and quantified by comparison to a standard for acetoacetyl-CoA orpeak area determined for 3-oxoadipyl-CoA (β-ketoadipyl-CoA).

FIG. 20 shows exemplary pathways from glutamate to hexamethylenediamine(HMDA) and 6-aminocaproate. The enzymes are designated as follows: A)glutamyl-CoA transferase and/or ligase, B) beta-ketothiolase, C)3-oxo-6-aminopimeloyl-CoA oxidoreductase, D)3-hydroxy-6-aminopimeloyl-CoA dehydratase, E)6-amino-7-carboxyhept-2-enoyl-CoA reductase, F) 6-aminopimeloyl-CoAreductase (aldehyde forming), G) 2-amino-7-oxoheptanoateaminotransferase and/or aminating oxidoreductase, H) homolysinedecarboxylase, I) 6-aminopimeloyl-CoA hydrolase, transferase and/orligase, J) 2-aminopimelate decarboxylase. The enzyme commission numberindicated for each reaction is described in Example XXVI below.

FIG. 21 shows exemplary pathways from glutaryl-CoA tohexamethylenediamine (HMDA) and 6-aminocaproate. The enzymes aredesignated as follows: A) glutaryl-CoA beta-ketothiolase, B)3-oxopimeloyl-CoA hydrolase, transferase and/or ligase, C) 3-oxopimelatereductase, D) 3-oxo-1-carboxyheptanal 7-aminotransferase and/or7-aminating oxidoreductase, E) 3-oxo-7-aminoheptanoate3-aminotransferase and/or 3-aminating oxidoreductase, F) 3-oxopimelatekinase, G) 5-oxopimeloylphosphonate reductase, H) 3-oxopimelate CoAtransferase and/or ligase, I) 5-oxopimeloyl-CoA reductase (aldehydeforming), J) 3-oxopimelate 3-aminotransferase and/or 3-aminatingoxidoreductase, K) 3-aminopimelate CoA transferase and/or ligase, L)5-aminopimeloyl-CoA reductase (aldehyde forming), M) 3-aminopimelatekinase, N) 5-aminopimeloylphosphonate reductase, 0) 3-aminopimelatereductase, P) 3-amino-7-oxoheptanoate 2,3-aminomutase, Q)2-amino-7-oxoheptanoate 7-aminotransferase and/or aminatingoxidoreductase, R) 3,7-diaminoheptanoate 2,3-aminomutase, S) homolysinedecarboxylase, T) 3-aminopimelate 2,3-aminomutase, U) 2-aminopimelatekinase, V) 2-aminopimelate CoA transferase and/or ligase, W)2-aminopimelate reductase, X) 6-aminopimeloylphosphonate reductase, Y)6-aminopimeloyl-CoA reductase (aldehyde forming), Z)3-amino-7-oxoheptanoate 7-aminotransferase and/or 7-aminatingoxidoreductase, AA) 2-aminopimelate decarboxylase and AB)3-oxo-1-carboxyheptanal 3-aminotransferase and/or 3-aminatingoxidoreductase. The enzyme commission number indicated for each reactionis described in XXVI below.

FIG. 22 shows an exemplary pathway from pyruvate and 4-aminobutanal tohexamethylenediamine (HMDA). The enzymes are designated as follows: A)2-oxo-4-hydroxy-7-aminoheptanoate aldolase, B)2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, C)2-oxo-7-aminohept-3-enoate reductase, D) 2-oxo-7-aminoheptanoateaminotransferase and/or aminating oxidoreductase, E) homolysinedecarboxylase, F) 2-oxo-7-aminoheptanoate decarboxylase, G)6-aminohexanal aminotransferase and/or 6-aminohexanal aminatingoxidoreductase. The enzyme commission number indicated for each reactionis described in Example XXVI below.

FIG. 23 shows an exemplary pathway from homolysine to 6-aminocaproate.Step A is catalyzed by homolysine 2-monooxygenase. Step B is hydrolysis,catalyzed by dilute acid or base.

FIG. 24 shows exemplary pathways from 6-aminocaproate tohexamethylenediamine. This figure depicts additional pathways further tothose presented in FIG. 13. The enzymes are designated as follows: A)6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproicsemialdehyde aminotransferase and/or 6-aminocaproic semialdehydeoxidoreductase (aminating), D) 6-aminocaproate N-acetyltransferase, E)6-acetamidohexanoate kinase, F) 6-AAHOP oxidoreductase, G)6-acetamidohexanal aminotransferase and/or 6-acetamidohexanaloxidoreductase (aminating), H) 6-acetamidohexanamine N-acetyltransferaseand/or 6-acetamidohexanamine hydrolase (amide), I) 6-acetamidohexanoateCoA transferase and/or 6-acetamidohexanoate CoA ligase, J)6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L)6-AHOP acyltransferase, M) 6-aminocaproate CoA transferase and/or6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoA oxidoreductase, 0)6-aminocaproate reductase and P) 6-acetamidohexanoate reductase.Abbreviations are: 6-AAHOP=[(6-acetamidohexanoyl)oxy]phosphonate and6-AHOP=[(6-aminohexanoyl)oxy]phosphonate. The enzyme commission numberindicated for each reaction is described in Example XXVI below.

FIG. 25 shows exemplary pathways from succinyl-CoA and acetyl-CoA tohexamethylenediamine (HMDA), caprolactam or levulinic acid. Pathways forthe production of adipate, 6-aminocaproate, caprolactam,hexamethylenediamine and levulinic acid from succinyl-CoA and acetyl-CoAare depicted. This figure depicts additional pathways further to thosepresented in FIG. 10. The enzymes are designated as follows: A)3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C)3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase,E) 3-oxoadipyl-CoA/acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G)3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I)3-hydroxyadipate dehydratase, J) 5-carboxy-2-pentenoate reductase, K)adipyl-CoA/acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoAhydrolase, N) adipyl-CoA reductase (aldehyde forming), 0)6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q)6-aminocaproyl-CoA/acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase,S) amidohydrolase, T) spontaneous cyclization, U) 6-aminocaproyl-CoAreductase (aldehyde forming), V) HMDA transaminase, W) HMDAdehydrogenase, X) adipate reductase, Y) adipate kinase, Z)adipylphosphate reductase, and AA) 3-oxoadipate decarboxylase.

FIG. 26 shows exemplary pathways from 2-amino-7-oxosubarate tohexamethylenediamine (HMDA) and 6-aminocaproate. The enzymes aredesignated as follows: A) 2-amino-7-oxosubarate keto-acid decarboxylase,B) 2-amino-7-oxoheptanoate decarboxylase, C) 6-aminohexanal aminatingoxidoreductase and/or 6-aminohexanal aminotransferase, D)2-amino-7-oxoheptanoate oxidoreductase, E) 2-aminopimelatedecarboxylase, F) 6-aminohexanal oxidoreductase, G)2-amino-7-oxoheptanoate decarboxylase, H) homolysine decarboxylase, I)2-amino-7-oxosubarate amino acid decarboxylase, J)2-oxo-7-aminoheptanoate aminating oxidoreductase and/or2-oxo-7-aminoheptanoate aminotransferase, K) 2-amino-7-oxosubarateaminating oxidoreductase and/or 2-amino-7-oxosubarate aminotransferase,L) 2,7-diaminosubarate decarboxylase and M) 2-amino-7-oxoheptanoateaminating oxidoreductase and/or 2-amino-7-oxoheptanoateaminotransferase. The enzyme commission number indicated for eachreaction is described in Example XXVI below.

FIG. 27 shows an exemplary pathway from glutamate-5-semialdehyde to2-amino-7-oxosubarate. The enzymes are designated as follows: A)2-amino-5-hydroxy-7-oxosubarate aldolase, B)2-amino-5-hydroxy-7-oxosubarate dehydratase, C)2-amino-5-ene-7-oxosubarate reductase.

FIG. 28 shows the 6-ACA versus growth yields for an ADHEr, LDH_D mutant.

FIG. 29 shows the 6-ACA versus growth yields for an ADHEr, MDH, ASPT,LDH_D mutant.

FIG. 30 shows the 6-ACA versus growth yields for an ADHEr, MDH, ASPT,LDH_D, THD2 mutant.

FIG. 31 shows the 6-ACA versus growth yields for an ADHEr, MDH, ASPT,LDH_D, GLUDy mutant.

FIG. 32 shows the 6-ACA versus growth yields for a PGI, EDA mutant or aPGI, PGDHy mutant.

FIG. 33 shows the 6-ACA versus growth yields for a PGI, EDA, ADHErmutant or a PGI, PGDHy, ADHEr mutant.

FIG. 34 shows the 6-ACA versus growth yields for an ADHEr, PGI, HEX1mutant.

FIG. 35 shows growth-coupled adipate production characteristics of highpriority strain designs (grey) compared with that of wild-type E. coli(black). A glucose uptake rate of 10 mmol/gDW/hr is assumed.

FIG. 36 shows the activity of CAR 889 and 891 using 20 mM Adipate.Activity is shown as units per mg of total protein in the crude lysate.

FIG. 37 shows the activity of CAR 720, 889, 890, 891 using 50 mM6-aminocaproate. Activity is shown as units per mg of total protein inthe crude lysate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities for6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid. The results described herein indicate that metabolic pathways canbe designed and recombinantly engineered to achieve the biosynthesis of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidin Escherichia coli and other cells or organisms. Biosyntheticproduction of 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid can be confirmed by construction of strains having thedesigned metabolic genotype. These metabolically engineered cells ororganisms also can be subjected to adaptive evolution to further augment6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidbiosynthesis, including under conditions approaching theoretical maximumgrowth.

As disclosed herein, a number of metabolic pathways for the productionof 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid are described. Two routes, the reverse adipate degradation pathwayand the 3-oxoadipate pathway, were found to be beneficial with respectto: (i) the adipate yields (92% molar yield on glucose), (ii) the lackof oxygen requirement for adipate synthesis, (iii) the associatedenergetics, and (iv) the theoretical capability to produce adipate asthe sole fermentation product. Metabolic pathways for adipate productionthat pass through α-ketoadipate or lysine are also described but arelower yielding and require aeration for maximum production. A pathwayfor producing either or both of 6-aminocaproate and caprolactam fromadipyl-CoA, a precursor in the reverse degradation pathway, is alsodisclosed herein.

As disclosed herein, a number of exemplary pathways for biosynthesis ofadipate are described. One exemplary pathway involves adipate synthesisvia a route that relies on the reversibility of adipate degradation asdescribed in organisms such as P. chrysogenum (see Examples I and II). Asecond exemplary pathway entails the formation of 3-oxoadipate followedby its reduction, dehydration and again reduction to form adipate (seeExamples III and IV). The adipate yield using either of these twopathways is 0.92 moles per mole glucose consumed. The uptake of oxygenis not required for attaining these theoretical maximum yields, and theenergetics under anaerobic conditions are favorable for growth andproduct secretion. A method for producing adipate from glucose-derivedcis,cis-muconic acid was described previously (Frost et al., U.S. Pat.No. 5,487,987, issued Jan. 30, 1996)(see Example V). Advantages of theembodiments disclosed herein over this previously described method arediscussed. Metabolic pathways for adipate production that pass throughα-ketoadipate (Example VI) or lysine (Example VII) precursors are loweryielding and require aeration for maximum production. A pathway forproducing either or both of 6-aminocaproate and caprolactam fromadipyl-CoA, a precursor in the reverse degradation pathway, is described(see Example VIII and IX). Additional pathways for producing adipate aredescribed in Examples X and XI. Pathways for producing any one, two,three or all four of 6-aminocaproate, caprolactam, hexamethylenediamineand levulinic acid from succinyl-CoA and acetyl-CoA are described inExamples XII, XXVIII. Several pathways for the production of6-aminocaproate from succinic semialdehyde and pyruvate are described inExample XIX. Several pathways for the production of hexamethylenediaminefrom 6-aminocaproate are described in Examples XX and XXVII. A pathwayfor producing either or both 6-aminocaproate and hexamethylenediaminefrom glutamate is described in Examples XXIV and XXV. Several pathwaysfor the production of hexamethylenediamine from glutaryl-CoA and atleast one pathway for production of 6-aminocaproate from glutaryl-CoAare described in Examples XXIV and XXV. A pathway for producing6-aminocaproate from homolysine is described in Example XXV. Pathwaysfor producing hexamethylenediamine from 2-amino-7-oxosubarate aredescribed in Example XXIV. Several pathways for producing6-aminocaproate are described in Example XXV. Exemplary genes andenzymes required for constructing microbes with these capabilities aredescribed as well as methods for cloning and transformation, monitoringproduct formation, and using the engineered microorganisms forproduction.

As disclosed herein, six different pathways for adipic acid synthesisusing glucose/sucrose as a carbon substrate are described. For allmaximum yield calculations, the missing reactions in a given pathwaywere added to the E. coli stoichiometric network in SimPheny that issimilar to the one described previously (Reed et al., Genome Biol. 4:R54(2003)). Adipate is a charged molecule under physiological conditionsand was assumed to require energy in the form of a proton-based symportsystem to be secreted out of the network. Such a transport system isthermodynamically feasible if the fermentations are carried out atneutral or near-neutral pH. Low pH adipic acid formation would requirean ATP-dependant export mechanism, for example, the ABC system asopposed to proton symport. The reactions in the pathways and methods ofimplementation of these pathways are described in Examples I-XI.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes within a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosyntheticpathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, “adipate,” having the chemical formula —OOC—(CH2)4-COO—(see FIG. 2) (IUPAC name hexanedioate), is the ionized form of adipicacid (IUPAC name hexanedioic acid), and it is understood that adipateand adipic acid can be used interchangeably throughout to refer to thecompound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled understand that thespecific form will depend on the pH.

As used herein, “6-aminocaproate,” having the chemical formula—OOC—(CH2)5-NH2 (see FIGS. 8 and 12), is the ionized form of6-aminocaproic acid (IUPAC name 6-aminohexanoic acid), and it isunderstood that 6-aminocaproate and 6-aminocaproic acid can be usedinterchangeably throughout to refer to the compound in any of itsneutral or ionized forms, including any salt forms thereof. It isunderstood by those skilled understand that the specific form willdepend on the pH.

As used herein, “caprolactam” (IUPAC name azepan-2-one) is a lactam of6-aminohexanoic acid (see FIG. 8).

As used herein, “hexamethylenediamine,” also referred to as1,6-diaminohexane or 1,6-hexanediamine, has the chemical formulaH2N(CH2)6NH2 (see FIGS. 10, 11 and 13).

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

As used herein, the term “osmoprotectant” when used in reference to aculture or growth condition is intended to mean a compound that acts asan osmolyte and helps a microbial organism as described herein surviveosmotic stress. Osmoprotectants include, for example, betaines, aminoacids, and the sugar trehalose. Non-limiting examples of such areglycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethyl sulfonio-2-methylproprionate,pipecolic acid, dimethyl sulfonioacetate, choline, L-carnitine andectoine.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical is intended to mean that the biosynthesis ofthe referenced biochemical is produced during the growth phase of amicroorganism. In a particular embodiment, the growth-coupled productioncan be obligatory, meaning that the biosynthesis of the referencedbiochemical is an obligatory product produced during the growth phase ofa microorganism.

As used herein, “metabolic modification” is intended to refer to abiochemical reaction that is altered from its naturally occurring state.Metabolic modifications can include, for example, elimination of abiochemical reaction activity by functional disruptions of one or moregenes encoding an enzyme participating in the reaction. Sets ofexemplary metabolic modifications are described herein (see ExampleXXX).

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive. The genetic alteration can be, forexample, deletion of the entire gene, deletion of a regulatory sequencerequired for transcription or translation, deletion of a portion of thegene which results in a truncated gene product, or by any of variousmutation strategies that inactivate the encoded gene product. Oneparticularly useful method of gene disruption is complete gene deletionbecause it reduces or eliminates the occurrence of genetic reversions inthe non-naturally occurring microorganisms of the invention.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

In the case of gene disruptions, a particularly useful stable geneticalteration is a gene deletion. The use of a gene deletion to introduce astable genetic alteration is particularly useful to reduce thelikelihood of a reversion to a phenotype prior to the geneticalteration. For example, stable growth-coupled production of abiochemical can be achieved, for example, by deletion of a gene encodingan enzyme catalyzing one or more reactions within a set of metabolicmodifications. The stability of growth-coupled production of abiochemical can be further enhanced through multiple deletions,significantly reducing the likelihood of multiple compensatoryreversions occurring for each disrupted activity.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes. In gene disruptionstrategies, evolutionally related genes can also be disrupted or deletedin a host microbial organism, paralogs or orthologs, to reduce oreliminate activities to ensure that any functional redundancy inenzymatic activities targeted for disruption do not short circuit thedesigned metabolic modifications.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

Disclosed herein are non-naturally occurring microbial organisms capableof producing adipate, 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid. For example, an adipate pathwaycan be a reverse adipate degradation pathway (see Examples I and II).For example, a non-naturally occurring microbial organism can have anadipate pathway including at least one exogenous nucleic acid encodingan adipate pathway enzyme expressed in a sufficient amount to produceadipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoAdehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoAsynthetase or phosphotransadipylase/adipate kinase oradipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. In addition,an adipate pathway can be through a 3-oxoadipate pathway (see ExamplesIII and IV). A non-naturally occurring microbial organism can have anadipate pathway including at least one exogenous nucleic acid encodingan adipate pathway enzyme expressed in a sufficient amount to produceadipate, the adipate pathway including succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase,3-hydroxyadipate dehydratase, and 2-enoate reductase.

Additionally, a non-naturally occurring microbial organism can have a6-aminocaproic acid pathway including at least one exogenous nucleicacid encoding a 6-aminocaproic acid pathway enzyme expressed in asufficient amount to produce 6-aminocaproic acid, the 6-aminocaproicacid pathway including CoA-dependent aldehyde dehydrogenase andtransaminase (see Examples VIII and IX). Alternatively, 6-aminocaproatedehydrogenase can be used to convert adipate semialdehyde to form6-aminocaproate (see FIG. 8). A non-naturally occurring microbialorganism can also have a caprolactam pathway including at least oneexogenous nucleic acid encoding a caprolactam pathway enzyme expressedin a sufficient amount to produce caprolactam, the caprolactam pathwayincluding CoA-dependent aldehyde dehydrogenase, transaminase or6-aminocaproate dehydrogenase, and amidohydrolase (see Examples VIII andIX).

As disclosed herein, a 6-aminocaproic acid or caprolactam producingmicrobial organism can produce 6-aminocaproic acid and/or caprolactamfrom an adipyl-CoA precursor (see FIG. 8 and Examples VIII and IX).Therefore, it is understood that a 6-aminocaproic acid or caprolactamproducing microbial organism can further include a pathway to produceadipyl-CoA. For example an adipyl-CoA pathway can include the enzymes ofFIG. 2 that utilize succinyl-CoA and acetyl-CoA as precursors throughthe production of adipyl-CoA, that is, lacking an enzyme for the finalstep of converting adipyl-CoA to adipate. Thus, one exemplary adipyl-CoApathway can include succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and5-carboxy-2-pentenoyl-CoA reductase.

In addition, as shown in FIG. 1, an adipate degradation pathway includesthe step of converting adipate to adipyl-CoA by an adipate CoA ligase.Therefore, an adipyl-CoA pathway can be an adipate pathway that furtherincludes an enzyme activity that converts adipate to adipyl-CoA,including, for example, adipate-CoA ligase activity as in the first stepof FIG. 1 or any of the enzymes in the final step of FIG. 2 carried outin the reverse direction, for example, any of adipyl-CoA synthetase(also referred to as adipate Co-A ligase), phosphotransadipylase/adipatekinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Anenzyme having adipate to adipyl-CoA activity can be an endogenousactivity or can be provided as an exogenous nucleic acid encoding theenzyme, as disclosed herein. Thus, it is understood that any adipatepathway can be utilized with an adipate to adipyl-CoA enzymatic activityto generate an adipyl-CoA pathway. Such a pathway can be included in a6-aminocaproic acid or caprolactam producing microbial organism toprovide an adipyl-CoA precursor for 6-aminocaproic acid and/orcaprolactam production.

An additional exemplary adipate pathway utilizes alpha-ketoadipate as aprecursor (see FIG. 6 and Example VI). For example, a non-naturallyoccurring microbial organism can have an adipate pathway including atleast one exogenous nucleic acid encoding an adipate pathway enzymeexpressed in a sufficient amount to produce adipate, the adipate pathwayincluding homocitrate synthase, homoaconitase, homoisocitratedehydrogenase, 2-ketoadipate reductase, alpha-hydroxyadipate dehydrataseand oxidoreductase. A further exemplary adipate pathway utilizes alysine dedgradation pathway (see FIG. 7 and Example VII). Anothernon-naturally occurring microbial organism can have an adipate pathwayincluding at least one exogenous nucleic acid encoding an adipatepathway enzyme expressed in a sufficient amount to produce adipate, theadipate pathway including carbon nitrogen lyase, oxidoreductase,transaminase and oxidoreductase.

Yet another exemplary adipate pathway utilizes alpha-ketoadipate as aprecursor (see FIG. 9 and Examples X and XI). Thus, a non-naturallyoccurring microbial organism can have an adipate pathway including atleast one exogenous nucleic acid encoding an adipate pathway enzymeexpressed in a sufficient amount to produce adipate, the adipate pathwayincluding alpha-ketoadipyl-CoA synthetase,phosphotransketoadipylase/alpha-ketoadipate kinase oralpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydrogenase; 2-hydroxyadipyl-CoA dehydratase;5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferaseor adipyl-CoA hydrolase. Additionally, a non-naturally occurringmicrobial organism can have an adipate pathway including at least oneexogenous nucleic acid encoding an adipate pathway enzyme expressed in asufficient amount to produce adipate, the adipate pathway including2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase,phosphotranshydroxyadipylase/2-hydroxyadipate kinase or2-hydroxyadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoAsynthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase.

As disclosed herein, the invention provides a non-naturally occurringmicrobial organism, including a microbial organism having a6-aminocaproic acid pathway including at least one exogenous nucleicacid encoding a 6-aminocaproic acid pathway enzyme expressed in asufficient amount to produce 6-aminocaproic acid, the 6-aminocaproicacid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase;3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoAdehydratase; 6-aminohex-2-enoyl-CoA reductase; and6-aminocaproyl-CoA/acyl-CoA transferase, 6-aminocaproyl-CoA synthase, or6-aminocaproyl-CoA hydrolase (see Examples XII and XIII; stepsA/B/C/D/K/L/M of FIG. 11). The invention additionally provides anon-naturally occurring microbial organism, including a microbialorganism having a 6-aminocaproic acid pathway including at least oneexogenous nucleic acid encoding a 6-aminocaproic acid pathway enzymeexpressed in a sufficient amount to produce 6-aminocaproic acid, the6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoAthiolase; 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase,3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoAhydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoatedehydratase; and 6-aminohex-2-enoate reductase (see Examples XII andXIV; steps A/E/F/G/H/I/J of FIG. 11).

In another embodiment, the invention provides a non-naturally occurringmicrobial organism, including a microbial organism having a caprolactampathway including at least one exogenous nucleic acid encoding acaprolactam pathway enzyme expressed in a sufficient amount to producecaprolactam, the caprolactam pathway including6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase(see Examples XII and XV; steps K/L of FIG. 11). Such a non-naturallyoccurring microbial organism containing a caprolactam pathway canfurther comprise a 6-aminocaproic acid pathway (see FIG. 11). Exemplary6-aminocaproic acid pathways include the 6-aminocaproic acid pathwayincluding CoA-dependent aldehyde dehydrogenase; and transaminase or6-aminocaproate dehydrogenase or the 6-aminocaproic acid pathwayincluding 3-oxo-6-aminohexanoyl-CoA thiolase;3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase,3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoAhydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoatedehydratase; and 6-aminohex-2-enoate reductase (steps A/E/F/G/H/I/J ofFIG. 11). It is understood that these or other exemplary 6-aminocaproicacid pathways disclosed herein can additionally be included in amicrobial organism having a caprolactam pathway, if desired. Theinvention also provides a non-naturally occurring microbial organism,including a microbial organism having a hexamethylenediamine pathwayincluding at least one exogenous nucleic acid encoding ahexamethylenediamine pathway enzyme expressed in a sufficient amount toproduce hexamethylenediamine, the hexamethylenediamine pathway including6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase;6-aminocaproyl-CoA reductase (aldehyde forming); andhexamethylenediamine transaminase or hexamethylenediamine dehydrogenase(see Example XII and XVI; steps K/L/N/O/P of FIG. 11). Such anon-naturally occurring microbial organism containing ahexamethylenediamine pathway can further comprise a 6-aminocaproic acidpathway (see FIG. 11). Exemplary 6-aminocaproic acid pathways includethe 6-aminocaproic acid pathway including CoA-dependent aldehydedehydrogenase; and transaminase or 6-aminocaproate dehydrogenase or the6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoAthiolase; 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase,3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoAhydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoatedehydratase; and 6-aminohex-2-enoate reductase (steps A/E/F/G/H/I/J ofFIG. 11). It is understood that these or other exemplary 6-aminocaproicacid pathways disclosed herein can additionally be included in amicrobial organism having a hexamethylenediamine pathway, if desired.

In yet another embodiment, the invention provides a non-naturallyoccurring microbial organism having a caprolactam pathway including atleast one exogenous nucleic acid encoding a caprolactam pathway enzymeexpressed in a sufficient amount to produce caprolactam, the caprolactampathway including 3-oxo-6-aminohexanoyl-CoA thiolase;3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoAdehydratase; and 6-aminohex-2-enoyl-CoA reductase (see Examples XII andXVII; steps A/B/C/D of FIG. 11). Also provided is a non-naturallyoccurring microbial organism having a hexamethylenediamine pathwayincluding at least one exogenous nucleic acid encoding ahexamethylenediamine pathway enzyme expressed in a sufficient amount toproduce hexamethylenediamine, the hexamethylenediamine pathway including3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase;3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoAreductase; 6-aminocaproyl-CoA reductase (aldehyde forming); andhexamethylenediamine transaminase or hexamethylenediamine dehydrogenase(see Examples XII and XVIII; steps A/B/C/D/N/O/P of FIG. 11).

In yet another embodiment, the invention provides a non-naturallyoccurring microbial organism having a 6-aminocaproic acid (6-ACA)pathway including at least one exogenous nucleic acid encoding a 6-ACApathway enzyme expressed in a sufficient amount to produce 6-ACA, the6-ACA pathway including a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH)aldolase, a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a2-oxohept-4-ene-1,7-dioate (OHED) reductase, a 2-oxoheptane-1,7-dioate(2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, anadipate semialdehyde oxidoreductase (aminating), a2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate(6-OHE) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) aminotransferase, a2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (aminating), a2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase, a2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (aminating), a2-aminohept-4-ene-1,7-dioate (2-AHE) reductase, a4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate-lyase, a4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, a3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA reductase, aadipyl-CoA dehydrogenase, a 2-oxohept-4-ene-1,7-dioate (OHED)formate-lyase, a 2-oxohept-4-ene-1,7-dioate (OHED) dehydrogenase, a2-oxoheptane-1,7-dioate (2-OHD) formate-lyase, a 2-oxoheptane-1,7-dioate(2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme(see Examples XIX and XXI; steps A-Q of FIG. 12). In a further aspect,the 6-ACA pathway includes a succinic semialdehyde dehydrogenase, analpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP)carboxykinase.

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHDdecarboxylase; or an adipate semialdehyde aminotransferase or an adipatesemialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; stepsA/B/C/D/E of FIG. 12). In a further aspect, the 6-ACA pathway includes asuccinic semialdehyde dehydrogenase, an alpha-ketoglutaratedecarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, wherein the set encodes an HODH aldolase; an OHED hydratase; anOHED reductase; a 2-OHD decarboxylase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating).

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHEreductase; or an adipate semialdehyde aminotransferase or an adipatesemialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; stepsA/B/F/G/E of FIG. 12). In a further aspect, the 6-ACA pathway includes asuccinic semialdehyde dehydrogenase, an alpha-ketoglutaratedecarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, where the set encode an HODH aldolase; an OHED hydratase; anOHED decarboxylase; a 6-OHE reductase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating).

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an OHED hydratase; an OHED aminotransferase or an OHEDoxidoreductase (aminating); a 2-AHE reductase; or a 2-AHD decarboxylase(see Examples XIX and XXI; steps A/B/J/D/I of FIG. 12). In a furtheraspect, the 6-ACA pathway includes a succinic semialdehydedehydrogenase, an alpha-ketoglutarate decarboxylase or aphosphoenolpyruvate (PEP) carboxykinase. In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding 6-ACA pathway enzymes, where the setencode an HODH aldolase; an OHED hydratase; an OHED aminotransferase oran OHED oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHDdecarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHDaminotransferase or a 2-OHD oxidoreductase (aminating); or a 2-AHDdecarboxylase (see Examples XIX and XXI; steps A/B/C/H/I of FIG. 12). Ina further aspect, the 6-ACA pathway includes a succinic semialdehydedehydrogenase, an alpha-ketoglutarate decarboxylase or aphosphoenolpyruvate (PEP) carboxykinase. In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding 6-ACA pathway enzymes, where the setencode an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHDaminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHDdecarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an HODH formate-lyase and a pyruvate formate-lyaseactivating enzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoAdehydratase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoAdehydrogenase; or an adipate semialdehyde aminotransferase or an adipatesemialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; stepsA/L/M/N/O/E of FIG. 12). In a further aspect, the 6-ACA pathway includesa succinic semialdehyde dehydrogenase, an alpha-ketoglutaratedecarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, where the set encode an HODH aldolase; an HODH formate-lyaseand a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase;a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; anadipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferaseor an adipate semialdehyde oxidoreductase (aminating).

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvateformate-lyase activating enzyme or OHED dehydrogenase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/P/N/O/Eof FIG. 12). In a further aspect, the 6-ACA pathway includes a succinicsemialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or aphosphoenolpyruvate (PEP) carboxykinase. In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding 6-ACA pathway enzymes, where the setencode an HODH aldolase; an OHED hydratase; an OHED formate-lyase and apyruvate formate-lyase activating enzyme or OHED dehydrogenase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating).

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anHODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHDformate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHDdehydrogenase; an adipyl-CoA dehydrogenase; or an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating)(see Examples XIX and XXI; steps A/B/C/Q/O/E of FIG. 12). In a furtheraspect, the 6-ACA pathway includes a succinic semialdehydedehydrogenase, an alpha-ketoglutarate decarboxylase or aphosphoenolpyruvate (PEP) carboxykinase. In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding 6-ACA pathway enzymes, where the setencode an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHDformate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHDdehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating).The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including aglutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an3-oxo-6-aminopimeloyl-CoA oxidoreductase, a3-hydroxy-6-aminopimeloyl-CoA dehydratase, a6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoAreductase (aldehyde forming), or a 2-aminopimelate decarboxylase (seeExamples XXV and XXVI; steps A/B/C/D/E/I/J of FIG. 20). In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, where the set encode a glutamyl-CoA transferase or glutamyl-CoAligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase;a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoAreductase (aldehyde forming); and a 2-aminopimelate decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase(see Examples XXV and XXVI; steps A/B/J/T/AA of FIG. 21). In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, where the set encode a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or3-oxopimelate aminating oxidoreductase; a 3-aminopimelate2,3-aminomutase; and a 2-aminopimelate decarboxylase. The inventionadditionally provides a non-naturally occurring microbial organismhaving a 6-aminocaproic acid (6-ACA) pathway including at least oneexogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in asufficient amount to produce 6-ACA, the 6-ACA pathway including ahomolysine 2-monooxygenase (see Examples XXV and XXVI; steps A of FIG.23). In a further aspect, the 6-ACA pathway includes hydrolysis of the6-aminohexanamide product by a dilute acid or base to convert6-aminohexanamide to 6-aminocaproate (see Examples XXV and XXVI; step Bof FIG. 23).

The invention additionally provides a non-naturally occurring microbialorganism having a 6-aminocaproic acid (6-ACA) pathway including at leastone exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed ina sufficient amount to produce 6-ACA, the 6-ACA pathway including anadipate reductase, an adipate kinase or an adipylphosphate reductase(see Example XXVIII; steps X/Y/Z of FIG. 25 and Example XXXI). In afurther aspect, the 6-ACA pathway includes an adipate reductase. Inanother further aspect, the 6-ACA pathway includes an adipate kinase andan adipylphosphate reductase. In still another aspect, the microbialorganism having the 6-aminocaproic acid (6-ACA) pathway above furthercomprises an adipate pathway, a caprolactam pathway and/or ahexamethylenediamine pathway described here (see Example XXVIII; stepsA-W of FIG. 25).

In one embodiment, the invention provides a non-naturally occurringmicrobial organism having a 6-aminocaproic acid (6-ACA) pathwayincluding at least one exogenous nucleic acid encoding a 6-ACA pathwayenzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACApathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a2-amino-7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoateoxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanaloxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV andXXVI; steps A/B/D/E/F/G/I of FIG. 26). In a further aspect, themicrobioal organism has a 2-amino-7-oxosubarate pathway having at leastone exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathwayenzyme expressed in a sufficient amount to produce2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase, a2-amino-5-hydroxy-7-oxosubarate dehydratase, or a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

In another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encoding6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of FIG. 26).In yet another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encoding6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of FIG. 26).In still yet another embodiment of the invention, the the non-naturallyoccurring microbial organism includes a set of exogenous nucleic acidsencoding 6-ACA pathway enzymes, where the set encodes a2-amino-7-oxosubarate amino acid decarboxylase; a2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanaloxidoreductase (see Example XXV; steps I/G/F of FIG. 26). In a furtheraspect of each of the above embodiments, the microbioal organism has a2-amino-7-oxosubarate pathway having a second set of exogenous nucleicacids encoding 2-amino-7-oxosubarate pathway enzymes expressed in asufficient amount to produce 2-amino-7-oxosubarate, the2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

In yet another embodiment, the invention provides a non-naturallyoccurring microbial organism having a hexamethylenediamine (HMDA)pathway including at least one exogenous nucleic acid encoding a HMDApathway enzyme expressed in a sufficient amount to produce HMDA, theHMDA pathway including a 6-aminocaproate kinase, an[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, a6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproicsemialdehyde oxidoreductase (aminating), a 6-aminocaproateN-acetyltransferase, a 6-acetamidohexanoate kinase, an[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase(aminating), a 6-acetamidohexanamine N-acetyltransferase, a6-acetamidohexanamine hydrolase (amide), a 6-acetamidohexanoate CoAtransferase, a 6-acetamidohexanoate CoA ligase, a6-acetamidohexanoyl-CoA oxidoreductase, a[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase, a[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase, a6-aminocaproate CoA transferase and a 6-aminocaproate CoA ligase (seeExamples XX and XXI; steps A-N of FIG. 13).

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a6-aminocaproate kinase; a 6-AHOP oxidoreductase; or a 6-aminocaproicsemialdehyde oxidoreductase (aminating) or a 6-aminocaproic acidsemialdehyde aminotransferase (see Examples XX and XXI; steps A/B/C ofFIG. 13). In another aspect of the invention, the non-naturallyoccurring microbial organism includes a set of exogenous nucleic acidsencoding HMDA pathway enzymes, wherein the set encodes a 6-aminocaproatekinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic semialdehydeoxidoreductase (aminating) or a 6-aminocaproic acid semialdehydeaminotransferase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoAoxidoreductase; or a 6-aminocaproic semialdehyde oxidoreductase(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (seeExamples XX and XXI; steps A/L/N/C of FIG. 13). In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding HMDA pathway enzymes, wherein theset encodes a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic semialdehydeoxidoreductase (aminating) or a 6-aminocaproic acid semialdehydeaminotransferase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a6-aminocaproate CoA transferase or a 6-aminocaproate CoA ligase; a6-aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic semialdehydeoxidoreductase (aminating) or a 6-aminocaproic acid semialdehydeaminotransferase (see Examples XX and XXI; steps M/N/C of FIG. 13). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a 6-aminocaproate CoA transferase or a6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a6-aminocaproic semialdehyde oxidoreductase (aminating) or a6-aminocaproic acid semialdehyde aminotransferase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); or a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide) (see Examples XX and XXI; steps D/E/F/G/H of FIG. 13).In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOPoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide).

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate CoAtransferase or a 6-acetamidohexanoate CoA ligase; a6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanalaminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); ora 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of FIG. 13).In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoAoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide). The invention additionally provides a non-naturallyoccurring microbial organism having a hexamethylenediamine (HMDA)pathway including at least one exogenous nucleic acid encoding a HMDApathway enzyme expressed in a sufficient amount to produce HMDA, theHMDA pathway including a 6-aminocaproate N-acetyltransferase; a6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a6-acetamidohexanal aminotransferase or a 6-acetamidohexanaloxidoreductase (aminating); or a 6-acetamidohexanamineN-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (seeExamples XX and XXI; steps D/E/K/J/G of FIG. 13). In another aspect ofthe invention, the non-naturally occurring microbial organism includes aset of exogenous nucleic acids encoding HMDA pathway enzymes, whereinthe set encodes a 6-aminocaproate N-acetyltransferase; a6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a6-acetamidohexanal aminotransferase or a 6-acetamidohexanaloxidoreductase (aminating); and a 6-acetamidohexanamineN-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). Theinvention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an3-oxo-6-aminopimeloyl-CoA oxidoreductase, a3-hydroxy-6-aminopimeloyl-CoA dehydratase, a6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoAreductase (aldehyde forming), a 2-amino-7-oxoheptanoateaminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, ora homolysine decarboxylase (see Examples XXIV and XXVI; steps A-H ofFIG. 20). In another aspect of the invention, the non-naturallyoccurring microbial organism includes a set of exogenous nucleic acidsencoding HMDA pathway enzymes, wherein the set encodes a glutamyl-CoAtransferase or ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoAoxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferaseor aminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a3-oxo-1-carboxyheptanal aminating oxidoreductase, a3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase, a 3-oxopimelate kinase, a5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming),a 3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelateligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ahomolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase(aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV andXXVI; FIG. 21).

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ora homolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/C/D/E/R/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 7-aminotransferase ora 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminatingoxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a5-oxopimeloyl-CoA reductase (aldehyde forming), a3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoAtransferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase(aldehyde forming); a 3-oxo-1-carboxyheptanal 7-aminotransferase or3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ora homolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/C/AB/Z/R/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase ora 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelatekinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of FIG. 21). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a5-oxopimeloyl-CoA reductase (aldehyde forming), a3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase,a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of FIG. 21). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehydeforming); a 3-oxo-1-carboxyheptanal 3-aminotransferase or a3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B//J/O/P/Q/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or a 3-oxopimelate aminating oxidoreductase; a3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonatereductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase (see ExamplesXXIV and XXVI; steps A/B/J/M/N/P/Q/S of FIG. 21). In another aspect ofthe invention, the non-naturally occurring microbial organism includes aset of exogenous nucleic acids encoding HMDA pathway enzymes, whereinthe set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonatereductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoAligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, ora homolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/J/K/L/P/Q/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelateCoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase;and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase,a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of FIG. 21). In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding HMDA pathway enzymes,wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoAligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoateaminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or ahomolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/J/K/L/Z/R/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelateCoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoateaminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonatereductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a3-amino-7-oxoheptanoate aminating oxidoreductase, a3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S of FIG. 21). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate7-aminotransferase or a 3-amino-7-oxoheptanoate aminatingoxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase (see ExamplesXXIV and XXVI; steps A/B/J/T/W/Q/S of FIG. 21). In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding HMDA pathway enzymes, wherein theset encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelatereductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelatekinase, a 6-aminopimeloylphosphonate reductase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase (see ExamplesXXIV and XXVI; steps A/B/J/T/U/X/Q/S of FIG. 21). In another aspect ofthe invention, the non-naturally occurring microbial organism includes aset of exogenous nucleic acids encoding HMDA pathway enzymes, whereinthe set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelatekinase; a 6-aminopimeloylphosphonate reductase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoAtransferase, 2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase(aldehyde forming), a 2-amino-7-oxoheptanoate 7-aminotransferase,2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or a 3-oxopimelate aminating oxidoreductase; a3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehydeforming); a 2-amino-7-oxoheptanoate 7-aminotransferase or2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoateaminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminatingoxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoatedecarboxylase, a 6-aminohexanal aminotransferase or a 6-aminohexanalaminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of FIG.22). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoateaminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;and a homolysine decarboxylase. In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes a2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoatedecarboxylase; and a 6-aminohexanal aminotransferase or a 6-aminohexanalaminating oxidoreductase.

The invention additionally provides a non-naturally occurring microbialorganism having a hexamethylenediamine (HMDA) pathway including at leastone exogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including a6-aminocaproate reductase, a 6-aminocaproic semialdehydeaminotransferase, a 6-aminocaproic semialdehyde oxidoreductase(aminating), 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoatereductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanaloxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase oracetamidohexanamine hydrolase (amide) (see Example XXVII; steps 0/C orD/P/G/H of FIG. 24 and Example XXXI). In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding HMDA pathway enzymes, wherein theset encodes a 6-aminocaproate reductase; and a 6-aminocaproicsemialdehyde aminotransferase or a 6-aminocaproic semialdehydeoxidoreductase (aminating). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate reductase;6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase(aminating); and 6-acetamidohexanamine N-acetyltransferase or6-acetamidohexanamine hydrolase (amide). The invention additionallyprovides a non-naturally occurring microbial organism having ahexamethylenediamine (HMDA) pathway including at least one exogenousnucleic acid encoding a HMDA pathway enzyme expressed in a sufficientamount to produce HMDA, the HMDA pathway including a2-amino-7-oxosubarate keto-acid decarboxylase, a 2-amino-7-oxoheptanoatedecarboxylase, a 6-aminohexanal aminating oxidoreductase, a6-aminohexanal aminotransferase, a 2-amino-7-oxoheptanoatedecarboxylase, a homolysine decarboxylase, a 2-amino-7-oxosubarate aminoacid decarboxylase, a 2-oxo-7-aminoheptanoate aminating oxidoreductase,a 2-oxo-7-aminoheptanoate aminotransferase, a 2-amino-7-oxosubarateaminating oxidoreductase, a 2-amino-7-oxosubarate aminotransferase, a2,7-diaminosubarate decarboxylase, a 2-amino-7-oxoheptanoateaminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase(see Examples XXIV and XXVI; Steps A/B/C/G/H/I/J/K/L/M of FIG. 26). In afurther aspect, the microbioal organism has a 2-amino-7-oxosubaratepathway having at least one exogenous nucleic acid encoding a2-amino-7-oxosubarate pathway enzyme expressed in a sufficient amount toproduce 2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathwayincluding a 2-amino-5-hydroxy-7-oxosubarate aldolase, a2-amino-5-hydroxy-7-oxosubarate dehydratase, or a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

In another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarateaminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (seeExamples XXIV and XXVI; steps K/L/H of FIG. 26). In another embodimentof the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding HMDA pathway enzymes,wherein the set encodes a 2-amino-7-oxosubarate amino aciddecarboxylase; a 2-oxo-7-aminoheptanoate aminating oxidoreductase or a2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase(see Examples XXIV and XXVI; steps I/J/H of FIG. 26). In anotherembodiment of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a 2-amino-7-oxosubarate amino aciddecarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a6-aminohexanal aminating oxidoreductase or a 6-aminohexanalaminotransferase (see Examples XXIV and XXVI; steps I/G/C of FIG. 26).In another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a6-aminohexanal aminating oxidoreductase or a 6-aminohexanalaminotransferase (see Examples XXIV and XXVI; steps A/B/C of FIG. 26).In another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate aminatingoxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and ahomolysine decarboxylase (see Examples XXIV and XXVI; steps A/M/H ofFIG. 26). In a further aspect of each of the above embodiments, themicrobioal organism has a 2-amino-7-oxosubarate pathway having a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27). The invention additionally provides a non-naturallyoccurring microbial organism having a levulinic acid (LA) pathwayincluding at least one exogenous nucleic acid encoding a LA pathwayenzyme expressed in a sufficient amount to produce LA, the LA pathwayincluding a 3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA/acyl-CoAtransferase, a 3-oxoadipyl-CoA synthase, a 3-oxoadipyl-CoA hydrolase, ora 3-oxoadipate decarboxylase (see Example XXIX; steps A/E/F/G/AA of FIG.25). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encoding LApathway enzymes, wherein the set encodes a 3-oxoadipyl-CoA thiolase; a3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, or a3-oxoadipyl-CoA hydrolase; and a 3-oxoadipate decarboxylase.

A non-naturally occurring microbial organism disclosed herein can have,for example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid pathway, wherein the non-naturally occurring microbialorganism includes at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product, as disclosed herein.Thus, a non-naturally occurring microbial organism can contain at leastone exogenous nucleic acid encoding a polypeptide, where the polypeptideis an enzyme or protein that converts the substrates and products of a6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway, such as that shown in FIGS. 2, 3, 8, 9, 10, 11, 12, 13 and20-27.

For example, a non-naturally occurring microbial organism can have anadipate pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from succinyl-CoA and acetyl-CoA to3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA;3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate (see FIG.2). Additionally, a non-naturally occurring microbial organism can havean adipate pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from succinyl-CoA and acetyl-CoA to3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; 3-oxoadipate to3-hydroxyadipate; 3-hydroxyadipate to hexa-2-enedioate (also referred toherein as 5-carboxy-2-pentenoate); hexa-2-enedioate to adipate (see FIG.3). Also, a non-naturally occurring microbial organism can have a6-aminocaproic acid pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from adipyl-CoA to adipate semialdehyde;and adipate semialdehyde to 6-aminocaproate (see FIG. 8). Furthermore, anon-naturally occurring microbial organism can have a caprolactampathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from adipyl-CoA to adipate semialdehyde; adipatesemialdehyde to 6-aminocaproate; and 6-aminocaproate to caprolactam.Additionally, a non-naturally occurring microbial organism can have anadipate pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from alpha-ketoadipate to alpha-ketoadipyl-CoA;alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; andadipyl-CoA to adipate (see FIG. 9). Also, a non-naturally occurringmicrobial organism can have an adipate pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromalpha-ketoadipate to 2-hydroxyadipate; 2-hydroxyadipate to2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate (FIG.9).

Additionally, a non-naturally occurring microbial organism can have a6-aminocaproyl-CoA pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from 4-aminobutyryl-CoA and acetyl-CoAto 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to3-hydroxy-6-aminohexanoyl-CoA; 3-hydroxy-6-aminohexanoyl-CoA to6-aminohex-2-enoyl-CoA; 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA(FIG. 11). Additional substrates and products of such a pathway caninclude 6-aminocaproyl-CoA to 6-aminocaproate; 6-aminocaproyl-CoA tocaprolactam; or 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde and6-aminocaproate semialdehyde to hexamethylenediamine (FIG. 11). Anon-naturally occurring microbial organism also can have a6-aminocaproic acid pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from 4-aminobutyryl-CoA and acetyl-CoAto 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to3-oxo-6-aminohexanoate; 3-oxo-6-aminohexanoate to3-hydroxy-6-aminohexanoate; 3-hydroxy-6-aminohexanoate to6-aminohex-2-enoate; and 6-aminohex-2-enoate to 6-aminocaproate (FIG.11). Additional substrates and products of such a pathway can include6-aminocaproate to caprolactam or 6-aminocaproate to 6-aminocaproyl-CoA,6-aminocaproyl-CoA to 6-aminocaproate semialdehyde, and 6-aminocaproatesemialdehyde to hexamethylenediamine (FIG. 11).

Additionally, a non-naturally occurring microbial organism can have a6-aminocaproic acid pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from pyruvate and succinic semialdehydeto 4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED): 2-oxohept-4-ene-1,7-dioate(OHED) to 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate(2-OHD) to adipate semialdehyde; and adipate semialdehyde to6-aminocaproate (FIG. 12). A non-naturally occurring microbial organismalternatively can have a 6-aminocaproic acid pathway, wherein themicrobial organism contains at least one exogenous nucleic acid encodinga polypeptide that converts a substrate to a product selected frompyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate;4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate(OHED); 2-oxohept-4-ene-1,7-dioate (OHED) to 6-oxohex-4-enoate (6-OHE):6-oxohex-4-enoate (6-OHE) to adipate semialdehyde; and adipatesemialdehyde to 6-aminocaproate (FIG. 12). A non-naturally occurringmicrobial organism alternatively can have a 6-aminocaproic acid pathway,wherein the microbial organism contains at least one exogenous nucleicacid encoding a polypeptide that converts a substrate to a productselected from pyruvate and succinic semialdehyde to4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate(OHED) to 2-aminohept-4-ene-1,7-dioate (2-AHE);2-aminohept-4-ene-1,7-dioate (2-AHE) to 2-aminoheptane-1,7-dioate(2-AHD); and 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproate (FIG.12). A non-naturally occurring microbial organism alternatively can havea 6-aminocaproic acid pathway, wherein the microbial organism containsat least one exogenous nucleic acid encoding a polypeptide that convertsa substrate to a product selected from pyruvate and succinicsemialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate;4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate(OHED); 2-oxohept-4-ene-1,7-dioate (OHED) to 2-oxoheptane-1,7-dioate(2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate(2-AHD); and 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproate (FIG.12). A non-naturally occurring microbial organism alternatively can havea 6-aminocaproic acid pathway, wherein the microbial organism containsat least one exogenous nucleic acid encoding a polypeptide that convertsa substrate to a product selected from pyruvate and succinicsemialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate;4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 3-hydroxyadipyl-CoA;3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA; 2,3-dehydroadipyl-CoA toadipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate semialdehydeto 6-aminocaproate (FIG. 12). A non-naturally occurring microbialorganism alternatively can have a 6-aminocaproic acid pathway, whereinthe microbial organism contains at least one exogenous nucleic acidencoding a polypeptide that converts a substrate to a product selectedfrom pyruvate and succinic semialdehyde to4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate(OHED) to 2,3-dehydroadipyl-CoA; 2,3-dehydroadipyl-CoA to adipyl-CoA;adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to6-aminocaproate (FIG. 12). A non-naturally occurring microbial organismalternatively can have a 6-aminocaproic acid pathway, wherein themicrobial organism contains at least one exogenous nucleic acid encodinga polypeptide that converts a substrate to a product selected frompyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate;4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate(OHED); 2-oxohept-4-ene-1,7-dioate (OHED) to 2-oxoheptane-1,7-dioate(2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to adipyl-CoA; adipyl-CoA toadipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (FIG.12).

Additionally, a non-naturally occurring microbial organism can have a6-aminocaproic acid pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from glutamate to glutamyl-CoA;glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl-CoAto 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoAto 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-aminopimelate; and2-aminopimelate to 6-aminocaproate (FIG. 20). A non-naturally occurringmicrobial organism alternatively can have a 6-aminocaproic acid pathway,wherein the microbial organism contains at least one exogenous nucleicacid encoding a polypeptide that converts a substrate to a productselected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (FIG. 21). Anon-naturally occurring microbial organism alternatively can have a6-aminocaproic acid pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from homolysine to 6-aminohexanamide;and 6-aminohexanamide to 6-aminocaproate (FIG. 23). A non-naturallyoccurring microbial organism alternatively can have a 6-aminocaproicacid pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from adipate to adipate semialdehyde; adipate toadipylphospate; and adipylphospate to adipate semialdehyde (FIG. 25).

Additionally, a non-naturally occurring microbial organism can have a6-aminocaproic acid pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from 2-amino-7-oxosubarate to2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal;6-aminohexanal to 6-aminocaproate; 2-amino-7-oxosubarate to2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal;2-amino-7-oxoheptanoate to 2-aminopimelate; and 2-aminopimelate to6-aminocaproate (FIG. 26). A non-naturally occurring microbial organismcan further have a 2-amino-7-oxosubarate pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromglutamate-5-semialdehyde to 2-amino-5-hydroxy-7-oxosubarate;2-amino-5-hydroxy-7-oxosubarate to 2-amino-5-ene-7-oxosubarate; and2-amino-5-ene-7-oxosubarate to 2-amino-7-oxosubarate (FIG. 27).Additionally, a non-naturally occurring microbial organism can have anhexamethylenediamine (HMDA) pathway, wherein the microbial organismcontains at least one exogenous nucleic acid encoding a polypeptide thatconverts a substrate to a product selected from 6-aminocaproate to[(6-aminohexanoyl)oxy]phosphonate (6-AHOP);[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproaicsemialdehyde; and 6-aminocaproaic semialdehyde to hexamethylenediamine(FIG. 13). A non-naturally occurring microbial organism alternativelycan have a HMDA pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from 6-aminocaproate to[(6-aminohexanoyl)oxy]phosphonate (6-AHOP);[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproyl-CoA;6-aminocaproyl-CoA to 6-aminocaproaic semialdehyde; and 6-aminocaproaicsemialdehyde to hexamethylenediamine (FIG. 13). A non-naturallyoccurring microbial organism alternatively can have a HMDA pathway,wherein the microbial organism contains at least one exogenous nucleicacid encoding a polypeptide that converts a substrate to a productselected from 6-aminocaproate to 6-aminocaproyl-CoA; 6-aminocaproyl-CoAto 6-aminocaproic semialdehyde; and 6-aminocaproic semialdehyde tohexamethylenediamine (FIG. 13). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected from6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP);[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP) to 6-acetamidohexanal;6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamineto hexamethylenediamine (FIG. 13). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected from6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to6-acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamidohexanal;6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamineto hexamethylenediamine (FIG. 13). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected from6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP);[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP) to6-acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamidohexanal;6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamineto hexamethylenediamine (FIG. 13).

Additionally, a non-naturally occurring microbial organism can have anhexamethylenediamine (HMDA) pathway, wherein the microbial organismcontains at least one exogenous nucleic acid encoding a polypeptide thatconverts a substrate to a product selected from glutamate toglutamyl-CoA; glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA;3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA;3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA;6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA;6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate; -amino-7-oxoheptanoateto homolysine; and homolysine to HMDA (FIG. 20). A non-naturallyoccurring microbial organism alternatively can have a HMDA pathway,wherein the microbial organism contains at least one exogenous nucleicacid encoding a polypeptide that converts a substrate to a productselected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to3-oxopimelate; 3-oxopimelate to 3-oxo-1-carboxy heptanal;3-oxo-1-carboxy heptanal to 3-oxo-7-amino heptanoate; 3-oxo-7-aminoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate tohomolysine; and homolysine to HMDA (FIG. 21). A non-naturally occurringmicrobial organism alternatively can have a HMDA pathway, wherein themicrobial organism contains at least one exogenous nucleic acid encodinga polypeptide that converts a substrate to a product selected fromglutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;3-oxopimelate to 5-oxopimeloyl phosponate; 5-oxopimeloyl phosponate to3-oxo-1-carboxy heptanal; 3-oxo-1-carboxy heptanal to 3-oxo-7-aminoheptanoate; 3-oxo-7-amino heptanoate to 3,7-diamino heptanoate;3,7-diamino heptanoate to homolysine and homolysine to HMDA (FIG. 21). Anon-naturally occurring microbial organism alternatively can have a HMDApathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA;5-oxopimeloyl-CoA to 3-oxo-1-carboxy heptanal; 3-oxo-1-carboxy heptanalto 3-oxo-7-amino heptanoate; 3-oxo-7-amino heptanoate to 3,7-diaminoheptanoate; 3,7-diamino heptanoate to homolysine and homolysine to HMDA(FIG. 21). A non-naturally occurring microbial organism alternativelycan have a HMDA pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-oxo-1-carboxyheptanal; 3-oxo-1-carboxy heptanal to 3-amino-7-oxoheptanoate;3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA (FIG. 21). Anon-naturally occurring microbial organism alternatively can have a HMDApathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA;5-oxopimeloyl-CoA to 3-oxo-1-carboxy heptanal; 3-oxo-1-carboxy heptanalto 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diaminoheptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA(FIG. 21). A non-naturally occurring microbial organism alternativelycan have a HMDA pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloylphosponate; 5-oxopimeloyl phosponate to 3-oxo-1carboxy heptanal;3-oxo-1-carboxy heptanal to 3-amino-7-oxoheptanoate;3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA (FIG. 21). Anon-naturally occurring microbial organism alternatively can have a HMDApathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate;3-aminopimelate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to2-amino-7-axoheptanoate; 2-amino-7-axoheptanoate to homolysine; andhomolysine to HMDA (FIG. 21). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromglutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 5-aminopimeloylphosphonate; 5-aminopimeloyl phosphonate to 3-amino-7-oxoheptanoate;3-amino-7-oxoheptanoate to 2-amino-7-axoheptanoate;2-amino-7-axoheptanoate to homolysine; and homolysine to HMDA (FIG. 21).A non-naturally occurring microbial organism alternatively can have aHMDA pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to5-aminopimeloyl-CoA; 5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoate;3-amino-7-oxoheptanoate to 2-amino-7-axoheptanoate;2-amino-7-axoheptanoate to homolysine; and homolysine to HMDA (FIG. 21).A non-naturally occurring microbial organism alternatively can have aHMDA pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate;3-aminopimelate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; andhomolysine to HMDA (FIG. 21). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromglutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;3-oxopimelate to 3-aminopimelate; 3-aminopimelate to5-aminopimeloyl-CoA; 5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoate;3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA (FIG. 21). Anon-naturally occurring microbial organism alternatively can have a HMDApathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate;3-aminopimelate to 5-aminopimeloyl phosphonate; 5-aminopimeloylphosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; andhomolysine to HMDA (FIG. 21). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromglutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate;2-aminopimelate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate tohomolysine; and homolysine to HMDA (FIG. 21). A non-naturally occurringmicrobial organism alternatively can have a HMDA pathway, wherein themicrobial organism contains at least one exogenous nucleic acid encodinga polypeptide that converts a substrate to a product selected fromglutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate;2-aminopimelate to 6-aminopimeloylphosphonate;6-aminopimeloylphosphonate to 2-amino-7-oxoheptanoate;2-amino-7-oxoheptanoate to homolysine; and homolysine to HMDA (FIG. 21).A non-naturally occurring microbial organism alternatively can have aHMDA pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate;3-aminopimelate to 2-aminopimelate; 2-aminopimelate to6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate;2-amino-7-oxoheptanoate to homolysine; and homolysine to HMDA (FIG. 21).A non-naturally occurring microbial organism alternatively can have aHMDA pathway, wherein the microbial organism contains at least oneexogenous nucleic acid encoding a polypeptide that converts a substrateto a product selected from pyruvate and 4-aminobutanal to2-oxo-4-hydroxy 7-aminoheptanoate; 2-oxo-4-hydroxy 7-aminoheptanoate to2-oxo-7-amino hept-3-enoate; 2-oxo-7-amino hept-3-enoate to2-oxo-7-amino heptanoate; 2-oxo-7-amino heptanoate to homolysine; andhomolysine to HMDA (FIG. 22). A non-naturally occurring microbialorganism alternatively can have a HMDA pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected frompyruvate and 4-aminobutanal to 2-oxo-4-hydroxy 7-aminoheptanoate;2-oxo-4-hydroxy 7-aminoheptanoate to 2-oxo-7-amino hept-3-enoate;2-oxo-7-amino hept-3-enoate to 2-oxo-7-amino heptanoate;2-oxo-7-aminoheptanoate to 6-aminohexanal; and 6-aminohexanal to HMDA(FIG. 22). A non-naturally occurring microbial organism alternativelycan have a HMDA pathway, wherein the microbial organism contains atleast one exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from 6-aminocaproate to 6-aminocaproicsemialdehyde; and 6-aminocaproic semialdehyde to HMDA (FIG. 24). Anon-naturally occurring microbial organism alternatively can have a HMDApathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from 6-aminocaproate to 6-acetamidohexanoate;6-acetamidohexanoate to 6-acetamidohexanal; 6-acetamidohexanal to6-acetamidohexanamine; 6-acetamidohexanamine to HMDA (FIG. 24). Anon-naturally occurring microbial organism alternatively can have a HMDApathway, wherein the microbial organism contains at least one exogenousnucleic acid encoding a polypeptide that converts a substrate to aproduct selected from 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate;2-amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to HMDA;2-amino-7-oxosubarate to 2-oxo-7-aminoheptanoate;2-amino-7-oxoheptanoate to homolysine; homolysine to HMDA;2-oxo-7-aminoheptanoate to homolysine; 2-oxo-7-aminoheptanoate to6-aminohexanal; 2-amino-7-oxosubarate to 2,7-diaminosubarate; and2,7-diaminosubarate to homolysine (FIG. 26). A non-naturally occurringmicrobial organism can further have a 2-amino-7-oxosubarate pathway,wherein the microbial organism contains at least one exogenous nucleicacid encoding a polypeptide that converts a substrate to a productselected from glutamate-5-semialdehyde to2-amino-5-hydroxy-7-oxosubarate; 2-amino-5-hydroxy-7-oxosubarate to2-amino-5-ene-7-oxosubarate; and 2-amino-5-ene-7-oxosubarate to2-amino-7-oxosubarate (FIG. 27).

Additionally, a non-naturally occurring microbial organism can have alevulinic acid pathway, wherein the microbial organism contains at leastone exogenous nucleic acid encoding a polypeptide that converts asubstrate to a product selected from succinyl-CoA and acetyl-CoA to3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; and 3-oxoadipate tolevulinic acid. It is understood that any of the pathways disclosedherein which produce an intermediate of one pathway can be used toproduce that intermediate for another pathway, if desired. For example,as disclosed herein, the alpha-ketoadipate to adipate pathway shown inFIG. 9 produces the intermediate adipyl-CoA, which is also anintermediate in the pathway depicted in FIG. 10. Thus, it is understoodthat an alternative pathway includes alpha-ketoadipate to adipyl-CoA,which can be converted to adipate, 6-aminocaporate, caprolactam orhexamethylenediamine, as depicted in FIG. 10. It is understood that anyof the pathways disclosed herein that produce a desired intermediate canbe used in combination with any other pathways disclosed herein so longas a desired product is produced. For example, a non-naturally occurringmicrobial organism disclosed herein, can have at least one nucleic acidencoding a 6-aminocaproic acid pathway enzyme and at least one nucleicacid encoding a hexamethylenediamine pathway enzyme, such as 2-AHDdecarboxylase (Step I of FIG. 12) and 6-acetamidohexanoate kinase (StepE of FIG. 13), or alternatively 2-oxohept-4-ene-1,7-dioate (OHED)decarboxylase (Step F of FIG. 12), adipate semialdehyde aminotransferase(Step E of FIG. 12) and 6-acetamidohexanoyl-CoA oxidoreductase (Step Jof FIG. 13), or alternatively 5-carboxy-2pentenoyl-CoA reductase (Step Dof FIG. 10), adipyl-CoA dehydrogenase (Step O of FIG. 12) and6-aminocaproyl-CoA oxidoreductase (Step N of FIG. 13), or alternatively2-amino-7-oxoheptanoate aminotransferase (Step G of FIG. 20) and3,7-diaminoheptanoate 2,3-aminomutase (Step R of FIG. 21), oralternatively 6-aminocaproate reductase (Step O of FIG. 24) and6-aminohex-2-enoate reductase (Step J of FIG. 11), or alternativelyadipate reductase (Step X of FIG. 25) and 6-acetamidohexanoate reductase(Step P of FIG. 24).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from any of the substrates or productsdisclosed herein or shown in any of FIGS. 1-14 and 20-27. One skilled inthe art will understand that any of the substrate-product pairsdisclosed herein suitable to produce a desired product and for which anappropriate activity is available for the conversion of the substrate tothe product can be readily determined by one skilled in the art based onthe teachings herein. Thus, the invention provides a non-naturallyoccurring microbial organism containing at least one exogenous nucleicacid encoding an enzyme or protein, where the enzyme or protein convertsthe substrates and products of a 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway, such as any of thoseshown in FIGS. 1-14 and 20-27.

While generally described herein as a microbial organism that contains a6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway, it is understood that the invention additionally provides anon-naturally occurring microbial organism comprising at least oneexogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway enzyme expressed in asufficient amount to produce an intermediate of a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid pathway. Forexample, as disclosed herein, 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway are exemplified in FIGS.1-14 and 20-27. Therefore, in addition to a microbial organismcontaining a 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid pathway that produces 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid, the invention additionallyprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid pathway enzyme,where the microbial organism produces a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid pathwayintermediate, for example, any of the intermediates shown in FIGS. 1-14and 20-27.

It is understood that any of the pathways disclosed herein, includingthose as described in the Examples and exemplified in the Figures,including the pathways of FIGS. 1-14 and 20-27, can be utilized togenerate a non-naturally occurring microbial organism that produces anypathway intermediate or product, as desired. As disclosed herein, such amicrobial organism that produces an intermediate can be used incombination with another microbial organism expressing downstreampathway enzymes to produce a desired product. However, it is understoodthat a non-naturally occurring microbial organism that produces a6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway intermediate can be utilized to produce the intermediate as adesired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction, and reference to anyof these metabolic constituents also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes as well as the reactantsand products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes participating in one or more 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosyntheticpathways. Depending on the host microbial organism chosen forbiosynthesis, nucleic acids for some or all of a particular6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidbiosynthetic pathway can be expressed. For example, if a chosen host isdeficient in one or more enzymes for a desired biosynthetic pathway,then expressible nucleic acids for the deficient enzyme(s) areintroduced into the host for subsequent exogenous expression.Alternatively, if the chosen host exhibits endogenous expression of somepathway genes, but is deficient in others, then an encoding nucleic acidis needed for the deficient enzyme(s) to achieve 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosynthesis. Thus,a non-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme activities to obtain a desiredbiosynthetic pathway or a desired biosynthetic pathway can be obtainedby introducing one or more exogenous enzyme activities that, togetherwith one or more endogenous enzymes, produces a desired product such as6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid.

Depending on the 6-aminocaproic acid, caprolactam, hexamethylenediamineor levulinic acid biosynthetic pathway constituents of a selected hostmicrobial organism, the non-naturally occurring microbial organisms ofthe invention will include at least one exogenously expressed6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway-encoding nucleic acid and up to all encoding nucleic acids forone or more adipate, 6-aminocaproic acid or caprolactam biosyntheticpathways. For example, 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthesis can be establishedin a host deficient in a pathway enzyme through exogenous expression ofthe corresponding encoding nucleic acid. In a host deficient in allenzymes of a 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid pathway, exogenous expression of all enzymes in thepathway can be included, although it is understood that all enzymes of apathway can be expressed even if the host contains at least one of thepathway enzymes.

For example, exogenous expression of all enzymes in a pathway forproduction of adipate can be included in a host organism, such assuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase orphosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase. In particular, a host organism cancontain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoAdehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoAsynthetase. Alternatively, a host organism can contain the adipatepathway enzymes succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and phosphotransadipylase/adipatekinase. In addition, a host organism can contain the adipate pathwayenzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA:acetyl-CoAtransferase. Further, a host organism can contain the adipate pathwayenzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA hydrolase.

In the case of a 6-aminocaproic acid producing microbial organism,exogenous expression of all enzymes in a pathway for production of6-aminocaproic acid can be included in a host organism, such asCoA-dependent aldehyde dehydrogenase and transaminase or CoA-dependentaldehyde dehydrogenase and 6-aminocaproate dehydrogenase. For acaprolactam producing microbial organism, exogenous expression of allenzymes in a pathway for production of caprolactam can be included in ahost organism, such as CoA-dependent aldehyde dehydrogenase,transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase. Inanother example, exogenous expression of all enzymes in a pathway forproduction of 6-aminocaproic acid (6-ACA) can be included in a hostorganism, such as an HODH aldolase; an OHED hydratase; an OHEDreductase; a 2-OHD decarboxylase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating),or alternatively an HODH aldolase; an OHED hydratase; an OHEDdecarboxylase; a 6-OHE reductase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating),or alternatively an HODH aldolase; an OHED hydratase; an OHEDaminotransferase or an OHED oxidoreductase (aminating); a 2-AHEreductase; and a 2-AHD decarboxylase, or alternatively an HODH aldolase;an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a2-OHD oxidoreductase (aminating); and a 2-AHD decarboxylase, oralternatively an HODH aldolase; an HODH formate-lyase and a pyruvateformate-lyase activating enzyme or an HODH dehydrogenase; a3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; anadipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferaseor an adipate semialdehyde oxidoreductase (aminating), or alternativelyan HODH aldolase; an OHED hydratase; an OHED formate-lyase and apyruvate formate-lyase activating enzyme or OHED dehydrogenase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating), or alternatively an HODH aldolase; an OHEDhydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvateformate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoAdehydrogenase; and an adipate semialdehyde aminotransferase or anadipate semialdehyde oxidoreductase (aminating). In a further aspect,all of the the 6-ACA pathway described above can include a succinicsemialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or aphosphoenolpyruvate (PEP) carboxykinase. In another example, exogenousexpression of all enzymes in a pathway for production of 6-aminocaproicacid (6-ACA) can be included in a host organism, such as a glutamyl-CoAtransferase or glutamyl-CoA ligase; a beta-ketothiolase; a3-oxo-6-aminopimeloyl-CoA oxidoreductase; a3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); and a 2-aminopimelate decarboxylase, oralternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelatedecarboxylase.

In another example, exogenous expression of all enzymes in a pathway forproduction of hexamethylenediamine can be included in a host organism,such as a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a6-aminocaproic semialdehyde oxidoreductase (aminating) or a6-aminocaproic acid semialdehyde aminotransferase, or alternatively a6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoAoxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase, oralternatively a 6-aminocaproate CoA transferase or a 6-aminocaproate CoAligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproicsemialdehyde oxidoreductase (aminating) or a 6-aminocaproic acidsemialdehyde aminotransferase, or alternatively a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOPoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide), or alternatively a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoAoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide), or alternatively a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOPoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide). In another example, exogenous expression of allenzymes in a pathway for production of hexamethylenediamine can beincluded in a host organism, such as a glutamyl-CoA transferase orligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase;a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferaseor aminating oxidoreductase; and a homolysine decarboxylase, oralternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminatingoxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a3-oxo-1-carboxyheptanal 7-aminotransferase or a 3-oxo-1-carboxyheptanal7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferaseor a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a5-oxopimeloyl-CoA reductase (aldehyde forming); a3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferaseor a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferaseor a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a5-oxopimeloyl-CoA reductase (aldehyde forming), a 5-oxopimeloyl-CoAhydrolase or a 5-oxopimeloyl-CoA ligase; a 3-oxo-1-carboxyheptanal3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoAtransferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoAreductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase;a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase, oralternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminatingoxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase, or alternatively a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or a 3-oxopimelate aminating oxidoreductase; a3-aminopimelate CoA transferase or a 3-aminopimelate CoA ligase; a5-aminopimeloyl-CoA reductase (aldehyde forming); a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoateaminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase, or alternatively a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or a 3-oxopimelate aminating oxidoreductase; a3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoateaminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase, or alternatively a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or 3-oxopimelate aminating oxidoreductase; a3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase, oralternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelatekinase; a 6-aminopimeloylphosphonate reductase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase, oralternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoAtransferase or 2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); a 2-amino-7-oxoheptanoate7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase;and a homolysine decarboxylase, or alternatively a2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoateaminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;and a homolysine decarboxylase, or alternatively a 6-aminocaproatereductase; and a 6-aminocaproic semialdehyde aminotransferase or a6-aminocaproic semialdehyde oxidoreductase (aminating), or alternativelya 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase;a 6-acetamidohexanal aminotransferase or 6-acetamidohexanaloxidoreductase (aminating); and a 6-acetamidohexanamineN-acetyltransferase or 6-acetamidohexanamine hydrolase (amide).

Depending on the 6-aminocaproic acid, caprolactam, hexamethylenediamineor levulinic acid biosynthetic pathway constituents of a selected hostmicrobial organism, the non-naturally occurring microbial organisms ofthe invention will include at least one exogenously expressed6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway-encoding nucleic acid and up to all encoding nucleic acids forone or more 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid biosynthetic pathways. For example, 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosynthesis can beestablished in a host deficient in a pathway enzyme or protein throughexogenous expression of the corresponding encoding nucleic acid. In ahost deficient in all enzymes or proteins of a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid pathway, exogenousexpression of all enzyme or proteins in the pathway can be included,although it is understood that all enzymes or proteins of a pathway canbe expressed even if the host contains at least one of the pathwayenzymes or proteins. For example, exogenous expression of all enzymes orproteins in a pathway for production of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid can be included, asdisclosed herein.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the adipate,6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveat least one, two, three, four, five, six, seven, eight, nine, ten,eleven or twelve, up to all nucleic acids encoding the above enzymesconstituting a 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid biosynthetic pathway. In some embodiments, thenon-naturally occurring microbial organisms also can include othergenetic modifications that facilitate or optimize 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosynthesis or thatconfer other useful functions onto the host microbial organism. One suchother functionality can include, for example, augmentation of thesynthesis of one or more of the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway precursors such assuccinyl-CoA and/or acetyl-CoA in the case of adipate synthesis, oradipyl-CoA or adipate in the case of 6-aminocaproic acid or caprolactamsynthesis, including the adipate pathway enzymes disclosed herein, orpyruvate and succinic semialdehyde, glutamate, glutaryl-CoA, homolysineor 2-amino-7-oxosubarate in the case of 6-aminocaprioate synthesis, or6-aminocaproate, glutamate, glutaryl-CoA, pyruvate and 4-aminobutanal,or 2-amino-7-oxosubarate in the case of hexamethylenediamine synthesis.

Generally, a host microbial organism is selected such that it producesthe precursor of a 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway, either as a naturallyproduced molecule or as an engineered product that either provides denovo production of a desired precursor or increased production of aprecursor naturally produced by the host microbial organism. A hostorganism can be engineered to increase production of a precursor, asdisclosed herein. In addition, a microbial organism that has beenengineered to produce a desired precursor can be used as a host organismand further engineered to express enzymes or proteins of a6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid. In this specific embodiment itcan be useful to increase the synthesis or accumulation of a6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway product to, for example, drive 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway reactions toward6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidproduction. Increased synthesis or accumulation can be accomplished by,for example, overexpression of nucleic acids encoding one or more of theabove-described 6-aminocaproic acid, caprolactam, hexamethylenediamineor levulinic acid pathway enzymes. Over expression of the 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid pathway enzymeor enzymes can occur, for example, through exogenous expression of theendogenous gene or genes, or through exogenous expression of theheterologous gene or genes. Therefore, naturally occurring organisms canbe readily generated to be non-naturally occurring microbial organismsof the invention, for example, producing 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid, throughoverexpression of at least one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, that is, up to allnucleic acids encoding 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthetic pathway enzymes. Inaddition, a non-naturally occurring organism can be generated bymutagenesis of an endogenous gene that results in an increase inactivity of an enzyme in the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

The invention additionally provides a non-naturally occurring microbialorganism that includes one or more gene disruptions, such as the genedisruptions disclosed in Example XXX and Tables 14-16, where theorganism produces a 6-ACA, adipate and/or HMDA. The disruptions occur ingenes encoding an enzyme that couples production of adipate, 6-ACAand/or HMDA to growth of the organism when the gene disruption reducesthe activity of the enzyme, such that the gene disruptions conferincreased production of adipate, 6-ACA and/or HMDA onto thenon-naturally occurring organism. Thus, the invention provides anon-naturally occurring microbial organism, comprising one or more genedisruptions, the one or more gene disruptions occurring in genesencoding proteins or enzymes wherein the one or more gene disruptionsconfer increased production of adipate, 6-ACA and/or HMDA in theorganism. As disclosed herein, such an organism contains a pathway forproduction of adipate, 6-ACA and/or HMDA, in addition to the genedisruptions, such as those exemplified in Example XXX and Tables 14-16.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid biosynthetic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosyntheticcapability. For example, a non-naturally occurring microbial organismhaving a 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid biosynthetic pathway can comprise at least two exogenousnucleic acids encoding desired enzymes. In the case of adipateproduction, at least two exogenous nucleic acids can encode the enzymessuch as the combination of succinyl-CoA:acetyl-CoA acyl transferase and3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyadipyl-CoA dehydratase, or 3-hydroxyadipyl-CoAand 5-carboxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA andadipyl-CoA synthetase, and the like. In the case of caprolactamproduction, at least two exogenous nucleic acids can encode the enzymessuch as the combination of CoA-dependent aldehyde dehydrogenase andtransaminase, or CoA-dependent aldehyde dehydrogenase andamidohydrolase, or transaminase and amidohydrolase. In the case of6-aminocaproic acid production, at least two exogenous nucleic acids canencode the enzymes such as the combination of an4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase and a2-oxohept-4-ene-1,7-dioate (OHED) hydratase, or a2-oxohept-4-ene-1,7-dioate (OHED) hydratase and a2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a 3-hydroxyadipyl-CoAdehydratase and a adipyl-CoA dehydrogenase, a glutamyl-CoA transferaseand a 6-aminopimeloyl-CoA hydrolase, or a glutaryl-CoA beta-ketothiolaseand a 3-aminopimelate 2,3-aminomutase. In the case ofhexamethylenediamine production, at least two exogenous nucleic acidscan encode the enzymes such as the combination of 6-aminocaproate kinaseand [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, or a6-acetamidohexanoate kinase and an [(6-acetamidohexanoyl)oxy]phosphonate(6-AAHOP) oxidoreductase, 6-aminocaproate N-acetyltransferase and6-acetamidohexanoyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoAdehydratase and a 2-amino-7-oxoheptanoate aminotransferase, or a3-oxopimeloyl-CoA ligase and a homolysine decarboxylase. Thus, it isunderstood that any combination of two or more enzymes of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention.

Similarly, it is understood that any combination of three or moreenzymes of a biosynthetic pathway can be included in a non-naturallyoccurring microbial organism of the invention, for example, in the caseof adipate production, the combination of enzymessuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, and 3-hydroxyadipyl-CoA dehydratase; orsuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase; orsuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase and adipyl-CoA synthetase; or 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase and adipyl-CoA:acetyl-CoAtransferase, and so forth, as desired, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product. In the case of 6-aminocaproic acidproduction, the at least three exogenous nucleic acids can encode theenzymes such as the combination of an 4-hydroxy-2-oxoheptane-1,7-dioate(HODH) aldolase, a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase and a2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, or a2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a2-aminohept-4-ene-1,7-dioate (2-AHE) reductase and a2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, or a3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase and aadipyl-CoA dehydrogenase, or a 6-amino-7-carboxyhept-2-enoyl-CoAreductase, a 6-aminopimeloyl-CoA hydrolase and a 2-aminopimelatedecarboxylase, or a glutaryl-CoA beta-ketothiolase, a 3-aminatingoxidoreductase and a 2-aminopimelate decarboxylase, or a 3-oxoadipyl-CoAthiolase, a 5-carboxy-2-pentenoate reductase and a adipate reductase. Inthe case of hexamethylenediamine production, at least three exogenousnucleic acids can encode the enzymes such as the combination of6-aminocaproate kinase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP)oxidoreductase and 6-aminocaproic semialdehyde aminotransferase, or a6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate kinase andan [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, or6-aminocaproate N-acetyltransferase, a[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase and6-acetamidohexanoyl-CoA oxidoreductase, or a 3-oxo-6-aminopimeloyl-CoAoxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase and ahomolysine decarboxylase, or a 2-oxo-4-hydroxy-7-aminoheptanoatealdolase, a 2-oxo-7-aminohept-3-enoate reductase and a homolysinedecarboxylase, or a 6-acetamidohexanoate reductase, a 6-acetamidohexanalaminotransferase and a 6-acetamidohexanamine N-acetyltransferase.Similarly, any combination of four or more enzymes of a biosyntheticpathway as disclosed herein can be included in a non-naturally occurringmicrobial organism of the invention, as desired, so long as thecombination of enzymes of the desired biosynthetic pathway results inproduction of the corresponding desired product.

In addition to the biosynthesis of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid other than use of the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid producers is through addition ofanother microbial organism capable of converting an adipate,6-aminocaproic acid or caprolactam pathway intermediate to6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid. One such procedure includes, for example, the fermentation of amicrobial organism that produces a 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway intermediate. The6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidpathway intermediate can then be used as a substrate for a secondmicrobial organism that converts the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway intermediate to6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid. The 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid pathway intermediate can be added directly to anotherculture of the second organism or the original culture of 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid pathwayintermediate producers can be depleted of these microbial organisms by,for example, cell separation, and then subsequent addition of the secondorganism to the fermentation broth can be utilized to produce the finalproduct without intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid. In theseembodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidcan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively,6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidalso can be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid intermediate and thesecond microbial organism converts the intermediate to 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid.

Similarly, it is understood by those skilled in the art that a hostorganism can be selected based on desired characteristics forintroduction of one or more gene disruptions to increase production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid. Thus, it is understood that, if a genetic modification is to beintroduced into a host organism to disrupt a gene, any homologs,orthologs or paralogs that catalyze similar, yet non-identical metabolicreactions can similarly be disrupted to ensure that a desired metabolicreaction is sufficiently disrupted. Because certain differences existamong metabolic networks between different organisms, those skilled inthe art will understand that the actual genes disrupted in a givenorganism may differ between organisms. However, given the teachings andguidance provided herein, those skilled in the art also will understandthat the methods of the invention can be applied to any suitable hostmicroorganism to identify the cognate metabolic alterations needed toconstruct an organism in a species of interest that will increase6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidbiosynthesis. In a particular embodiment, the increased productioncouples biosynthesis of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid to growth of the organism, andcan obligatorily couple production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid to growth of the organism ifdesired and as disclosed herein.

Sources of encoding nucleic acids for a 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid pathway enzyme caninclude, for example, any species where the encoded gene product iscapable of catalyzing the referenced reaction. Such species include bothprokaryotic and eukaryotic organisms including, but not limited to,bacteria, including archaea and eubacteria, and eukaryotes, includingyeast, plant, insect, animal, and mammal, including human. Exemplaryspecies for such sources include, for example, Escherichia coli,Escherichia coli str. K12, Escherichia coli C, Escherichia coli W,Pseudomonas sp, Pseudomonas knackmussii, Pseudomonas sp. Strain B13,Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri,Pseudomonas mendocina, Rhodopseudomonas palustris, Mycobacteriumtuberculosis, Vibrio cholera, Heliobacter pylori, Klebsiella pneumoniae,Serratia proteamaculans, Streptomyces sp. 2065, Pseudomonas aeruginosa,Pseudomonas aeruginosa PA01, Ralstonia eutropha, Ralstonia eutropha H16,Clostridium acetobutylicum, Euglena gracilis, Treponema denticola,Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp.ADP1, Acinetobacter sp. Strain M-1, Streptomyces coelicolor, Eubacteriumbarkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum,Clostridium botulinum A3 str, Clostridium tyrobutyricum, Clostridiumpasteurianum, Clostridium thermoaceticum (Moorella thermoaceticum),Moorella thermoacetica Acinetobacter calcoaceticus, Mus musculus, Susscrofa, Flavobacterium sp, Arthrobacter aurescens, Penicilliumchrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis,Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimiasucciniciproducens, Clostridium ljungdahlii, Clostridiumcarboxydivorans, Geobacillus stearothermophilus, Agrobacteriumtumefaciens, Achromobacter denitrificans, Arabidopsis thaliana,Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp.M62/1, Fusobacterium nucleatum, Bos taurus, Zoogloea ramigera,Rhodobacter sphaeroides, Clostridium beijerinckii, Metallosphaerasedula, Thermoanaerobacter species, Thermoanaerobacter brockii,Acinetobacter baylyi, Porphyromonas gingivalis, Leuconostocmesenteroides, Sulfolobus tokodaii, Sulfolobus tokodaii 7, Sulfolobussolfataricus, Sulfolobus solfataricus, Sulfolobus acidocaldarius,Salmonella typhimurium, Salmonella enterica, Thermotoga maritima,Halobacterium salinarum, Bacillus cereus, Clostridium difficile,Alkaliphilus metalliredigenes, Thermoanaerobacter tengcongensis,Saccharomyces kluyveri, Helicobacter pylori, Corynebacterium glutamicum,Clostridium saccharoperbutylacetonicum, Pseudomonas chlororaphis,Streptomyces clavuligerus, Campylobacter jejuni, Thermus thermophilus,Pelotomaculum thermopropionicum, Bacteroides capillosus, Anaerotruncuscolihominis, Natranaerobius thermophilius, Archaeoglobus fulgidus,Archaeoglobus fulgidus DSM 4304, Haloarcula marismortui, Pyrobaculumaerophilum, Pyrobaculum aerophilum str. IM2, Nicotiana tabacum, Menthepiperita, Pinus taeda, Hordeum vulgare, Zea mays, Rhodococcus opacus,Cupriavidus necator, Bradyrhizobium japonicum, Bradyrhizobium japonicumUSDA 110, Ascarius suum, butyrate-producing bacterium L2-50, Bacillusmegaterium, Methanococcus maripaludis, Methanosarcina mazei,Methanosarcina mazei, Methanocarcina barkeri, Methanocaldococcusjannaschii, Caenorhabditis elegans, Leishmania major, Methylomicrobiumalcaliphilum 20Z, Chromohalobacter salexigens, Archaeglubus fulgidus,Chlamydomonas reinhardtii, Trichomonas vaginalis G3, Trypanosoma brucei,Mycoplana ramose, Micrococcus luteas, Acetobacter pasteurians,Kluyveromyces lactis, Mesorhizobium loti, Lactococcus lactis,Lysinibacillus sphaericus, Candida boidinii, Candida albicans SC5314,Burkholderia ambifaria AMMD, Ascaris suun, Acinetobacter baumanii,Acinetobacter calcoaceticus, Burkholderia phymatum, Candida albicans,Clostridium subterminale, Cupriavidus taiwanensis, Flavobacteriumlutescens, Lachancea kluyveri, Lactobacillus sp. 30a, Leptospirainterrogans, Moorella thermoacetica, Myxococcus xanthus, Nicotianaglutinosa, Nocardia iowensis (sp. NRRL 5646), Pseudomonas reinekei MT1,Ralstonia eutropha JMP 134, Ralstonia metallidurans, Rhodococcus jostii,Schizosaccharomyces pombe, Selenomonas ruminantium, Streptomycesclavuligenus, Syntrophus aciditrophicus, Vibrio parahaemolyticus, Vibriovulnificus, as well as other exemplary species disclosed herein oravailable as source organisms for corresponding genes (see Examples).However, with the complete genome sequence available for now more than550 species (with more than half of these available on public databasessuch as the NCBI), including 395 microorganism genomes and a variety ofyeast, fungi, plant, and mammalian genomes, the identification of genesencoding the requisite 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthetic activity for one ormore genes in related or distant species, including for example,homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic alterations enabling biosynthesis of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid described hereinwith reference to a particular organism such as E. coli can be readilyapplied to other microorganisms, including prokaryotic and eukaryoticorganisms alike. Given the teachings and guidance provided herein, thoseskilled in the art will know that a metabolic alteration exemplified inone organism can be applied equally to other organisms.

In some instances, such as when a 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthetic pathway exists in anunrelated species, 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthesis can be conferredonto the host species by, for example, exogenous expression of a paralogor paralogs from the unrelated species that catalyzes a similar, yetnon-identical metabolic reaction to replace the referenced reaction.Because certain differences among metabolic networks exist betweendifferent organisms, those skilled in the art will understand that theactual gene usage between different organisms may differ. However, giventhe teachings and guidance provided herein, those skilled in the artalso will understand that the teachings and methods of the invention canbe applied to all microbial organisms using the cognate metabolicalterations to those exemplified herein to construct a microbialorganism in a species of interest that will synthesize 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizopus oryzae, and the like. For example,E. coli is a particularly useful host organisms since it is a wellcharacterized microbial organism suitable for genetic engineering. Otherparticularly useful host organisms include yeast such as Saccharomycescerevisiae. It is understood that any suitable microbial host organismcan be used to introduce metabolic and/or genetic modifications toproduce a desired product.

Methods for constructing and testing the expression levels of anon-naturally occurring 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid-producing host can be performed,for example, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidcan be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid biosynthetic pathway encoding nucleic acids as exemplified hereinoperably linked to expression control sequences functional in the hostorganism. Expression vectors applicable for use in the microbial hostorganisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Additionally, the expression vectorscan include one or more selectable marker genes and appropriateexpression control sequences. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art. Such methods include, for example, nucleic acid analysissuch as Northern blots or polymerase chain reaction (PCR) amplificationof mRNA, or immunoblotting for expression of gene products, or othersuitable analytical methods to test the expression of an introducednucleic acid sequence or its corresponding gene product. It isunderstood by those skilled in the art that the exogenous nucleic acidis expressed in a sufficient amount to produce the desired product, andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art and asdisclosed herein.

Directed evolution is one approach that involves the introduction ofmutations targeted to a specific gene in order to improve and/or alterthe properties of an enzyme. Improved and/or altered enzymes can beidentified through implementation screening assays that allow for theidentification of useful variants. Particularly useful screening methodsinclude sensitive high-throughput assays that allow the automatedscreening of many enzyme variants (e.g., >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to identify an enzymewith optimized properties. The greater the number of variants screened,the higher the probability of identifying an ideally suitable variant.Computational algorithms that can help to identify areas of the gene formutagenesis also have been developed and can significantly reduce thenumber of enzyme variants that need to be generated and screened.

Numerous directed evolution technologies have been developed (forreviews, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman andLalonde, In Biocatalysis in the pharmaceutical and biotechnologyindustries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax.Biomol. Eng 22:1-9 (2005).; and Sen et al., Appl Biochem. Biotechnol143:212-223 (2007)) to be effective at creating diverse variantlibraries and these methods have been successfully applied to theimprovement of a wide range of properties across many enzyme classes.

Enzyme characteristics that have been improved and/or altered bydirected evolution technologies include, for example,selectivity/specificity—for conversion of non-natural substrates;temperature stability—for robust high temperature processing; pHstability—for bioprocessing under lower or higher pH conditions;substrate or product tolerance—so that high product titers can beachieved; binding (K_(m))—broadens substrate binding to includenon-natural substrates; inhibition (K_(i))—to remove inhibition byproducts, substrates, or key intermediates; activity (kcat)—increasesenzymatic reaction rates to achieve desired flux; expressionlevels—increases protein yields and overall pathway flux; oxygenstability—for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity—for operation of an aerobic enzyme inthe absence of oxygen.

The following exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Any of these can be used to alter/optimize activity of adecarboxylase enzyme.

EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005)) introducesrandom point mutations by reducing the fidelity of DNA polymerase in PCRreactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations,or by other conditional variations. The five step cloning process toconfine the mutagenesis to the target gene of interest involves: 1)error-prone PCR amplification of the gene of interest; 2) restrictionenzyme digestion; 3) gel purification of the desired DNA fragment; 4)ligation into a vector; 5) transformation of the gene variants into asuitable host and screening of the library for improved performance.This method can generate multiple mutations in a single genesimultaneously, which can be useful. A high number of mutants can begenerated by EpPCR, so a high-throughput screening assay or a selectionmethod (especially using robotics) is useful to identify those withdesirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., NucleicAcids Res 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497(2006)) has many of the same elements as epPCR except a whole circularplasmid is used as the template and random 6-mers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats. Adjusting the Mn²⁺concentration can vary the mutation rate somewhat. This technique uses asimple error-prone, single-step method to create a full copy of theplasmid with 3-4 mutations/kbp. No restriction enzyme digestion orspecific primers are required. Additionally, this method is typicallyavailable as a kit.

DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci U.S.A.91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)) typicallyinvolves digestion of two or more variant genes with nucleases such asDnase I or EndoV to generate a pool of random fragments that arereassembled by cycles of annealing and extension in the presence of DNApolymerase to create a library of chimeric genes. Fragments prime eachother and recombination occurs when one copy primes another copy(template switch). This method can be used with >1 kbp DNA sequences. Inaddition to mutational recombinants created by fragment reassembly, thismethod introduces point mutations in the extension steps at a ratesimilar to error-prone PCR. The method can be used to removedeleterious, random and neutral mutations that might conferantigenicity.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol 16:258-261(1998)) entails template priming followed by repeated cycles of 2 stepPCR with denaturation and very short duration of annealing/extension (asshort as 5 sec). Growing fragments anneal to different templates andextend further, which is repeated until full-length sequences are made.Template switching means most resulting fragments have multiple parents.Combinations of low-fidelity polymerases (Taq and Mutazyme) reduceerror-prone biases because of opposite mutational spectra.

In Random Priming Recombination (RPR) random sequence primers are usedto generate many short DNA fragments complementary to different segmentsof the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Basemisincorporation and mispriming via epPCR give point mutations. ShortDNA fragments prime one another based on homology and are recombined andreassembled into full-length by repeated thermocycling. Removal oftemplates prior to this step assures low parental recombinants. Thismethod, like most others, can be performed over multiple iterations toevolve distinct properties. This technology avoids sequence bias, isindependent of gene length, and requires very little parent DNA for theapplication.

In Heteroduplex Recombination linearized plasmid DNA is used to formheteroduplexes that are repaired by mismatch repair. (Volkov et al,Nucleic Acids Res 27:e18 (1999); and Volkov et al., Methods Enzymol.328:456-463 (2000)) The mismatch repair step is at least somewhatmutagenic. Heteroduplexes transform more efficiently than linearhomoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al.,Nat. Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation andsize fractionation of ssDNA. Homologous fragments are hybridized in theabsence of polymerase to a complementary ssDNA scaffold. Any overlappingunhybridized fragment ends are trimmed down by an exonuclease. Gapsbetween fragments are filled in, and then ligated to give a pool offull-length diverse strands hybridized to the scaffold (that contains Uto preclude amplification). The scaffold then is destroyed and isreplaced by a new strand complementary to the diverse strand by PCRamplification. The method involves one strand (scaffold) that is fromonly one parent while the priming fragments derive from other genes; theparent scaffold is selected against. Thus, no reannealing with parentalfragments occurs. Overlapping fragments are trimmed with an exonuclease.Otherwise, this is conceptually similar to DNA shuffling and StEP.Therefore, there should be no siblings, few inactives, and no unshuffledparentals. This technique has advantages in that few or no parentalgenes are created and many more crossovers can result relative tostandard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails templateswitching of unidirectionally growing strands from primers in thepresence of unidirectional ssDNA fragments used as a pool of templates.(Lee et al., J. Molec. Catalysis 26:119-129 (2003)) No DNA endonucleasesare used. Unidirectional ssDNA is made by DNA polymerase with randomprimers or serial deletion with exonuclease. Unidirectional ssDNA areonly templates and not primers. Random priming and exonucleases don'tintroduce sequence bias as true of enzymatic cleavage of DNAshuffling/RACHITT. RETT can be easier to optimize than StEP because ituses normal PCR conditions instead of very short extensions.Recombination occurs as a component of the PCR steps—no directshuffling. This method can also be more random than StEP due to theabsence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primersare used to control recombination between molecules; (Bergquist andGibbs, Methods Mol. Biol 352:191-204 (2007); Bergquist et al., Biomol.Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can beused to control the tendency of other methods such as DNA shuffling toregenerate parental genes. This method can be combined with randommutagenesis (epPCR) of selected gene segments. This can be a good methodto block the reformation of parental sequences. No endonucleases areneeded. By adjusting input concentrations of segments made, one can biastowards a desired backbone. This method allows DNA shuffling fromunrelated parents without restriction enzyme digests and allows a choiceof random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)creates a combinatorial library with 1 base pair deletions of a gene orgene fragment of interest. (Ostermeier et al., Proc Natl Acad Sci U.S.A.96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol 17:1205-1209(1999)) Truncations are introduced in opposite direction on pieces of 2different genes. These are ligated together and the fusions are cloned.This technique does not require homology between the 2 parental genes.When ITCHY is combined with DNA shuffling, the system is called SCRATCHY(see below). A major advantage of both is no need for homology betweenparental genes; for example, functional fusions between an E. coli and ahuman gene were created via ITCHY. When ITCHY libraries are made, allpossible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs areused to generate truncations. (Lutz et al., Nucleic Acids Res 29:E16(2001)) Relative to ITCHY, THIO-ITCHY can be easier to optimize, providemore reproducibility, and adjustability.

SCRATCHY combines two methods for recombining genes, ITCHY and DNAshuffling. (Lutz et al., Proc Natl Acad Sci U.S.A. 98:11248-11253(2001)) SCRATCHY combines the best features of ITCHY and DNA shuffling.First, ITCHY is used to create a comprehensive set of fusions betweenfragments of genes in a DNA homology-independent fashion. Thisartificial family is then subjected to a DNA-shuffling step to augmentthe number of crossovers. Computational predictions can be used inoptimization. SCRATCHY is more effective than DNA shuffling whensequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed byscreening/selection for those retaining usable activity. (Bergquist etal., Biomol. Eng 22:63-72 (2005)) Then, these are used in DOGS togenerate recombinants with fusions between multiple active mutants orbetween active mutants and some other desirable parent. Designed topromote isolation of neutral mutations; its purpose is to screen forretained catalytic activity whether or not this activity is higher orlower than in the original gene. RNDM is usable in high throughputassays when screening is capable of detecting activity above background.RNDM has been used as a front end to DOGS in generating diversity. Thetechnique imposes a requirement for activity prior to shuffling or othersubsequent steps; neutral drift libraries are indicated to result inhigher/quicker improvements in activity from smaller libraries. Thoughpublished using epPCR, this could be applied to other large-scalemutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis methodthat: 1) generates pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage; this pool isused as a template to 2) extend in the presence of “universal” basessuch as inosine; 3) replication of a inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis. (Wong et al.,Biotechnol J 3:74-82 (2008); Wong et al., Nucleic Acids Res 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)) Using thistechnique it can be possible to generate a large library of mutantswithin 2-3 days using simple methods. This technique is non-directed incomparison to the mutational bias of DNA polymerases. Differences inthis approach makes this technique complementary (or an alternative) toepPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed toencode “all genetic diversity in targets” and allow a very highdiversity for the shuffled progeny. (Ness et al., Nat. Biotechnol20:1251-1255 (2002)) In this technique, one can design the fragments tobe shuffled. This aids in increasing the resulting diversity of theprogeny. One can design sequence/codon biases to make more distantlyrelated sequences recombine at rates approaching those observed withmore closely related sequences. Additionally, the technique does notrequire physically possessing the template genes.

Nucleotide Exchange and Excision Technology NexT exploits a combinationof dUTP incorporation followed by treatment with uracil DNA glycosylaseand then piperidine to perform endpoint DNA fragmentation. (Muller etal., Nucleic Acids Res 33:e117 (2005)) The gene is reassembled usinginternal PCR primer extension with proofreading polymerase. The sizesfor shuffling are directly controllable using varying dUPT::dTTP ratios.This is an end point reaction using simple methods for uracilincorporation and cleavage. Other nucleotide analogs, such as8-oxo-guanine, can be used with this method. Additionally, the techniqueworks well with very short fragments (86 bp) and has a low error rate.The chemical cleavage of DNA used in this technique results in very fewunshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC) alinker is used to facilitate fusion between two distantly/unrelatedgenes. Nuclease treatment is used to generate a range of chimerasbetween the two genes. These fusions result in libraries ofsingle-crossover hybrids. (Sieber et al., Nat. Biotechnol 19:456-460(2001)) This produces a limited type of shuffling and a separate processis required for mutagenesis. In addition, since no homology is neededthis technique can create a library of chimeras with varying fractionsof each of the two unrelated parent genes. SHIPREC was tested with aheme-binding domain of a bacterial CP450 fused to N-terminal regions ofa mammalian CP450; this produced mammalian activity in a more solubleenzyme.

In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials area supercoiled dsDNA plasmid containing an insert and two primers whichare degenerate at the desired site of mutations. (Kretz et al., MethodsEnzymol. 388:3-11 (2004)) Primers carrying the mutation of interest,anneal to the same sequence on opposite strands of DNA. The mutation istypically in the middle of the primer and flanked on each side by ˜20nucleotides of correct sequence. The sequence in the primer is NNN orNNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A, C). Afterextension, DpnI is used to digest dam-methylated DNA to eliminate thewild-type template. This technique explores all possible amino acidsubstitutions at a given locus (i.e., one codon). The techniquefacilitates the generation of all possible replacements at a single-sitewith no nonsense codons and results in equal to near-equalrepresentation of most possible alleles. This technique does not requireprior knowledge of the structure, mechanism, or domains of the targetenzyme. If followed by shuffling or Gene Reassembly, this technologycreates a diverse library of recombinants containing all possiblecombinations of single-site up-mutations. The utility of this technologycombination has been demonstrated for the successful evolution of over50 different enzymes, and also for more than one property in a givenenzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of shortoligonucleotide cassettes to replace limited regions with a large numberof possible amino acid sequence alterations. (Reidhaar-Olson et al.Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science241:53-57 (1988)) Simultaneous substitutions at two or three sites arepossible using this technique. Additionally, the method tests a largemultiplicity of possible sequence changes at a limited range of sites.This technique has been used to explore the information content of thelambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentiallysimilar to CCM except it is employed as part of a larger program: 1) Useof epPCR at high mutation rate to 2) ID hot spots and hot regions andthen 3) extension by CMCM to cover a defined region of protein sequencespace. (Reetz, M. T., S. Wilensek, D. Zha, and K. E. Jaeger, 2001,Directed Evolution of an Enantioselective Enzyme through CombinatorialMultiple-Cassette Mutagenesis. Angew. Chem. Int. Ed Engl. 40:3589-3591.)As with CCM, this method can test virtually all possible alterationsover a target region. If used along with methods to create randommutations and shuffled genes, it provides an excellent means ofgenerating diverse, shuffled proteins. This approach was successful inincreasing, by 51-fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique conditional ts mutator plasmids allowincreases of 20- to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required. (Selifonova et al., Appl Environ Microbiol67:3645-3649 (2001)) This technology is based on a plasmid-derived mutD5gene, which encodes a mutant subunit of DNA polymerase III. This subunitbinds to endogenous DNA polymerase III and compromises the proofreadingability of polymerase III in any strain that harbors the plasmid. Abroad-spectrum of base substitutions and frameshift mutations occur. Inorder for effective use, the mutator plasmid should be removed once thedesired phenotype is achieved; this is accomplished through atemperature sensitive origin of replication, which allows for plasmidcuring at 41° C. It should be noted that mutator strains have beenexplored for quite some time (e.g., see Low et al., J. Mol. Biol.260:359-3680 (1996)). In this technique very high spontaneous mutationrates are observed. The conditional property minimizes non-desiredbackground mutations. This technology could be combined with adaptiveevolution to enhance mutagenesis rates and more rapidly achieve desiredphenotypes.

“Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis methodthat assesses and optimizes combinatorial mutations of selected aminoacids.” (Rajpal et al., Proc Natl Acad Sci U.S.A. 102:8466-8471 (2005))Rather than saturating each site with all possible amino acid changes, aset of nine is chosen to cover the range of amino acid R-groupchemistry. Fewer changes per site allows multiple sites to be subjectedto this type of mutagenesis. A >800-fold increase in binding affinityfor an antibody from low nanomolar to picomolar has been achievedthrough this method. This method is a rational approach to minimize thenumber of random combinations and can increase the ability to findimproved traits by greatly decreasing the numbers of clones to bescreened. This has been applied to antibody engineering, specifically toincrease the binding affinity and/or reduce dissociation. The techniquecan be combined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied tomultiple genes at one time or to creating a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation). Typically this technologyis used in combination with ultra-high-throughput screening to query therepresented sequence space for desired improvements. This techniqueallows multiple gene recombinations independent of homology. The exactnumber and position of cross-over events can be pre-determined usingfragments designed via bioinformatic analysis. This technology leads toa very high level of diversity with virtually no parental genereformation and a low level of inactive genes. Combined with GSSM™, alarge range of mutations can be tested for improved activity. The methodallows “blending” and “fine tuning” of DNA shuffling, e.g. codon usagecan be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithmthat anchors the structurally defined protein backbone possessing aparticular fold, and searches sequence space for amino acidsubstitutions that can stabilize the fold and overall proteinenergetics. (Hayes et al., Proc Natl Acad Sci U.S.A. 99:15926-15931(2002)) This technology uses in silico structure-based entropypredictions in order to search for structural tolerance toward proteinamino acid variations. Statistical mechanics is applied to calculatecoupling interactions at each position. Structural tolerance towardamino acid substitution is a measure of coupling. Ultimately, thistechnology is designed to yield desired modifications of proteinproperties while maintaining the integrity of structuralcharacteristics. The method computationally assesses and allowsfiltering of a very large number of possible sequence variants (10⁵⁰).The choice of sequence variants to test is related to predictions basedon the most favorable thermodynamics. Ostensibly only stability orproperties that are linked to stability can be effectively addressedwith this technology. The method has been successfully used in sometherapeutic proteins, especially in engineering immunoglobulins. Insilico predictions avoid testing extraordinarily large numbers ofpotential variants. Predictions based on existing three-dimensionalstructures are more likely to succeed than predictions based onhypothetical structures. This technology can readily predict and allowtargeted screening of multiple simultaneous mutations, something notpossible with purely experimental technologies due to exponentialincreases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) use knowledge ofstructure/function to choose a likely site for enzyme improvement; 2)saturation mutagenesis at chosen site using Stratagene QuikChange (orother suitable means); 3) screen/select for desired properties; and 4)with improved clone(s), start over at another site and continuerepeating. (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz etal., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)) This is a provenmethodology, which assures all possible replacements at a given positionare made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques.

The invention additionally provides methods for producing a desiredintermediate or product such as adipate, 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid. For example, amethod for producing adipate can involve culturing a non-naturallyoccurring microbial organism having an adipate pathway, the pathwayincluding at least one exogenous nucleic acid encoding an adipatepathway enzyme expressed in a sufficient amount to produce adipate,under conditions and for a sufficient period of time to produce adipate,the adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase orphosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase. Additionally, a method forproducing adipate can involve culturing a non-naturally occurringmicrobial organism having an adipate pathway, the pathway including atleast one exogenous nucleic acid encoding an adipate pathway enzymeexpressed in a sufficient amount to produce adipate, under conditionsand for a sufficient period of time to produce adipate, the adipatepathway including succinyl-CoA:acetyl-CoA acyl transferase,3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipatedehydratase, and 2-enoate reductase.

Further, a method for producing 6-aminocaproic acid can involveculturing a non-naturally occurring microbial organism having a6-aminocaproic acid pathway, the pathway including at least oneexogenous nucleic acid encoding a 6-aminocaproic acid pathway enzymeexpressed in a sufficient amount to produce 6-aminocaproic acid, underconditions and for a sufficient period of time to produce 6-aminocaproicacid, the 6-aminocaproic acid pathway including CoA-dependent aldehydedehydrogenase and transaminase or 6-aminocaproate dehydrogenase.Additionally, a method for producing caprolactam can involve culturing anon-naturally occurring microbial organism having a caprolactam pathway,the pathway including at least one exogenous nucleic acid encoding acaprolactam pathway enzyme expressed in a sufficient amount to producecaprolactam, under conditions and for a sufficient period of time toproduce caprolactam, the caprolactam pathway including CoA-dependentaldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase,and amidohydrolase.

The invention additionally provides methods for producing 6-aminocaproicacid (6-ACA) by culturing a non-naturally occurring microbial organismhaving a 6-ACA pathway described herein under conditions and for asufficient period of time to produce 6-ACA. In one aspect the 6-ACApathway includes an HODH aldolase; an OHED hydratase; an OHED reductase;a 2-OHD decarboxylase; and an adipate semialdehyde aminotransferase oran adipate semialdehyde oxidoreductase (aminating). In another aspect,the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHEDdecarboxylase; a 6-OHE reductase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating).In yet another aspect, the 6-ACA pathway includes an HODH aldolase; anOHED hydratase; an OHED aminotransferase or an OHED oxidoreductase(aminating); a 2-AHE reductase; and a 2-AHD decarboxylase. In still yetanother aspect, the 6-ACA pathway includes an HODH aldolase; an OHEDhydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHDoxidoreductase (aminating); and a 2-AHD decarboxylase. In still yetanother aspect, the 6-ACA pathway includes an HODH aldolase; an HODHformate-lyase and a pyruvate formate-lyase activating enzyme or an HODHdehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating). In still yet another aspect, the 6-ACApathway includes an HODH aldolase; an OHED hydratase; an OHEDformate-lyase and a pyruvate formate-lyase activating enzyme or OHEDdehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoAdehydrogenase; and an adipate semialdehyde aminotransferase or anadipate semialdehyde oxidoreductase (aminating). In still yet anotheraspect, the 6-ACA pathway includes an HODH aldolase; an OHED hydratase;an OHED reductase; a 2-OHD formate-lyase and a pyruvate formate-lyaseactivating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA dehydrogenase;and an adipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating). In a further aspect, the 6-ACA pathwaysdescribed above can include a succinic semialdehyde dehydrogenase, analpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP)carboxykinase.

The invention additionally provides methods for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway described herein underconditions and for a sufficient period of time to produce HMDA. In oneaspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOPoxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase. Inanother aspect, the HMDA pathway includes a 6-aminocaproate kinase; a6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a6-aminocaproic semialdehyde oxidoreductase (aminating) or a6-aminocaproic acid semialdehyde aminotransferase. In yet anotheraspect, the HMDA pathway includes a 6-aminocaproate CoA transferase or a6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a6-aminocaproic semialdehyde oxidoreductase (aminating) or a6-aminocaproic acid semialdehyde aminotransferase. In still yet anotheraspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase;a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a6-acetamidohexanal aminotransferase or a 6-acetamidohexanaloxidoreductase (aminating); and a 6-acetamidohexanamineN-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). Instill yet another aspect, the HMDA pathway includes a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoAoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide). In still yet another aspect, the HMDA pathwayincludes a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoatekinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferaseor a 6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide).

Also, a method for producing adipate can involve culturing anon-naturally occurring microbial organism having an adipate pathway,the pathway including at least one exogenous nucleic acid encoding anadipate pathway enzyme expressed in a sufficient amount to produceadipate, under conditions and for a sufficient period of time to produceadipate, the adipate pathway including alpha-ketoadipyl-CoA synthetase,phosphotransketoadipylase/alpha-ketoadipate kinase oralpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydrogenase; 2-hydroxyadipyl-CoA dehydratase;5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferaseor adipyl-CoA hydrolase. Furthermore, a method for producing adipate caninvolve culturing a non-naturally occurring microbial organism having anadipate pathway, the pathway including at least one exogenous nucleicacid encoding an adipate pathway enzyme expressed in a sufficient amountto produce adipate, under conditions and for a sufficient period of timeto produce adipate, the adipate pathway including 2-hydroxyadipatedehydrogenase; 2-hydroxyadipyl-CoA synthetase,phosphotranshydroxyadipylase/2-hydroxyadipate kinase or2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoAsynthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase.

As disclosed herein, the invention also provides method for producing6-aminocaproic acid by culturing a non-naturally occurring microbialorganism having a 6-aminocaproic acid pathway including at least oneexogenous nucleic acid encoding a 6-aminocaproic acid pathway enzymeexpressed in a sufficient amount to produce 6-aminocaproic acid, the6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoAthiolase; 3-oxo-6-aminohexanoyl-CoA reductase;3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoAreductase; and 6-aminocaproyl-CoA/acyl-CoA transferase,6-aminocaproyl-CoA synthase, or 6-aminocaproyl-CoA hydrolase (seeExamples XII and XIII; steps A/B/C/D/K/L/M of FIG. 11). The inventionadditionally provides a method for producing 6-aminocaproic acid byculturing a non-naturally occurring microbial organism having a6-aminocaproic acid pathway including at least one exogenous nucleicacid encoding a 6-aminocaproic acid pathway enzyme expressed in asufficient amount to produce 6-aminocaproic acid, the 6-aminocaproicacid pathway including 3-oxo-6-aminohexanoyl-CoA thiolase;3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase,3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoAhydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoatedehydratase; and 6-aminohex-2-enoate reductase (see Examples XII andXIV; steps A/E/F/G/H/I/J of FIG. 11).

In another embodiment, the invention provides a method for producingcaprolactam by culturing a non-naturally occurring microbial organismhaving a caprolactam pathway including at least one exogenous nucleicacid encoding a caprolactam pathway enzyme expressed in a sufficientamount to produce caprolactam, the caprolactam pathway including6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase(see Examples XII and XV; steps K/L of FIG. 11). In such a method, thecaprolactam can be produced by spontaneous cyclization of6-aminocaproyl-CoA to caprolactam (see Example XII; step Q of FIG. 11).The invention also provides a non-naturally occurring microbial organismhaving a hexamethylenediamine pathway including at least one exogenousnucleic acid encoding a hexamethylenediamine pathway enzyme expressed ina sufficient amount to produce hexamethylenediamine, thehexamethylenediamine pathway including 6-aminocaproyl-CoA/acyl-CoAtransferase or 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase(aldehyde forming); and hexamethylenediamine transaminase orhexamethylenediamine dehydrogenase (see Examples XII and XVI; stepsK/L/N/O/P of FIG. 11).

In yet another embodiment, the invention provides a method for producingcaprolactam by culturing a non-naturally occurring microbial organismhaving a caprolactam pathway including at least one exogenous nucleicacid encoding a caprolactam pathway enzyme expressed in a sufficientamount to produce caprolactam, the caprolactam pathway including3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase;3-hydroxy-6-aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-CoAreductase (see Examples XII and XVII; steps A/B/C/D of FIG. 11). In sucha method, the caprolactam can be produced by spontaneous cyclization of6-aminocaproyl-CoA to caprolactam (see Example XII; step Q of FIG. 11).Also provided is a method for producing hexamethylenediamine byculturing a non-naturally occurring microbial organism having ahexamethylenediamine pathway including at least one exogenous nucleicacid encoding a hexamethylenediamine pathway enzyme expressed in asufficient amount to produce hexamethylenediamine, thehexamethylenediamine pathway including 3-oxo-6-aminohexanoyl-CoAthiolase; 3-oxo-6-aminohexanoyl-CoA reductase;3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoAreductase; 6-aminocaproyl-CoA reductase (aldehyde forming); andhexamethylenediamine transaminase or hexamethylenediamine dehydrogenase(see Examples XII and XVIII; steps A/B/C/D/N/O/P of FIG. 11).

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-ACA pathway, the microbial organismincluding at least one exogenous nucleic acid encoding a 6-ACA pathwayenzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACApathway including a succinic semialdehyde dehydrogenase, analpha-ketoglutarate decarboxylase, a phosphoenolpyruvate (PEP)carboxykinase, a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, a2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a2-oxohept-4-ene-1,7-dioate (OHED) reductase, a 2-oxoheptane-1,7-dioate(2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, anadipate semialdehyde oxidoreductase (aminating), a2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate(6-OHE) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) aminotransferase, a2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (aminating), a2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase, a2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (aminating), a2-aminohept-4-ene-1,7-dioate (2-AHE) reductase, a4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate-lyase, a4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, a3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA reductase, aadipyl-CoA dehydrogenase, a 2-oxohept-4-ene-1,7-dioate (OHED)formate-lyase, a 2-oxohept-4-ene-1,7-dioate (OHED) dehydrogenase, a2-oxoheptane-1,7-dioate (2-OHD) formate-lyase, a 2-oxoheptane-1,7-dioate(2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme(see Examples XIX and XXI; steps A-Q of FIG. 12).

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-ACA pathway, the microbial organismincluding at least one exogenous nucleic acid encoding a 6-ACA pathwayenzyme expressed in a sufficient amount to produce 6-ACA. In one aspectthe 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an OHEDreductase; a 2-OHD decarboxylase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating)(see Examples XIX and XXI; steps A/B/C/D/E of FIG. 12). In anotheraspect of the invention, the 6-ACA pathway includes an HODH aldolase; anOHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipatesemialdehyde aminotransferase or an adipate semialdehyde oxidoreductase(aminating) (see Examples XIX and XXI; steps A/B/F/G/E of FIG. 12). Inanother aspect of the invention, the 6-ACA pathway includes an HODHaldolase; an OHED hydratase; an OHED aminotransferase or an OHEDoxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase(see Examples XIX and XXI; steps A/B/J/D/I of FIG. 12). In anotheraspect of the invention, the 6-ACA pathway includes an HODH aldolase; anOHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHDoxidoreductase (aminating); and a 2-AHD decarboxylase (see Examples XIXand XXI; steps A/B/C/H/I of FIG. 12). In another aspect of theinvention, the 6-ACA pathway includes an HODH aldolase; an HODHformate-lyase and a pyruvate formate-lyase activating enzyme or an HODHdehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating) (see Examples XIX and XXI; steps A/L/M/N/O/Eof FIG. 12). the 6-ACA pathway includes an HODH aldolase; an OHEDhydratase; an OHED formate-lyase and a pyruvate formate-lyase activatingenzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; anadipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferaseor an adipate semialdehyde oxidoreductase (aminating) (see Examples XIXand XXI; steps A/B/P/N/O/E of FIG. 12). In another aspect of theinvention, the 6-ACA pathway includes an HODH aldolase; an OHEDhydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvateformate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoAdehydrogenase; and an adipate semialdehyde aminotransferase or anadipate semialdehyde oxidoreductase (aminating) (see Examples XIX andXXI; steps A/B/C/Q/O/E of FIG. 12). In a further aspect, the 6-ACApathways described above can include a succinic semialdehydedehydrogenase, an alpha-ketoglutarate decarboxylase or aphosphoenolpyruvate (PEP) carboxykinase.

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-ACA pathway including at least oneexogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in asufficient amount to produce 6-ACA, the 6-ACA pathway including aglutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an3-oxo-6-aminopimeloyl-CoA oxidoreductase, a3-hydroxy-6-aminopimeloyl-CoA dehydratase, a6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoAreductase (aldehyde forming), or a 2-aminopimelate decarboxylase (seeExamples XXV and XXVI; steps A/B/C/D/E/I/J of FIG. 20). In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, where the set encode a glutamyl-CoA transferase or glutamyl-CoAligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase;a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); and a 2-aminopimelate decarboxylase.

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-ACA pathway including at least oneexogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in asufficient amount to produce 6-ACA, the 6-ACA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase(see Examples XXV and XXVI; steps A/B/J/T/AA of FIG. 21). In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding 6-ACA pathwayenzymes, where the set encode a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or3-oxopimelate aminating oxidoreductase; a 3-aminopimelate2,3-aminomutase; and a 2-aminopimelate decarboxylase.

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-ACA pathway including at least oneexogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in asufficient amount to produce 6-ACA, the 6-ACA pathway including ahomolysine 2-monooxygenase (see Examples XXV and XXVI; steps A of FIG.23). In a further aspect, the 6-ACA pathway includes hydrolysis of the6-aminohexanamide product by a dilute acid or base to convert6-aminohexanamide to 6-aminocaproate (see Example XXV; steps B of FIG.23).

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-ACA pathway including at least oneexogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in asufficient amount to produce 6-ACA, the 6-ACA pathway including anadipate reductase, an adipate kinase or an adipylphosphate reductase(see Example XXVIII; steps X/Y/Z of FIG. 25). In a further aspect, the6-ACA pathway includes an adipate reductase. In another further aspect,the 6-ACA pathway includes an adipate kinase and an adipylphosphatereductase. In still another aspect, the microbial organism having the6-aminocaproic acid (6-ACA) pathway above further comprises an adipatepathway, a caprolactam pathway and/or a hexamethylenediamine pathwaydescribed here (see Example XXVIII; steps A-W of FIG. 25).

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid (6-ACA) by culturing a non-naturally occurringmicrobial organism having a 6-aminocaproic acid (6-ACA) pathwayincluding at least one exogenous nucleic acid encoding a 6-ACA pathwayenzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACApathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a2-amino-7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoateoxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanaloxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV andXXVI; steps A/B/D/E/F/G/I of FIG. 26). In a further aspect, themicrobioal organism has a 2-amino-7-oxosubarate pathway having at leastone exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathwayenzyme expressed in a sufficient amount to produce2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase, a2-amino-5-hydroxy-7-oxosubarate dehydratase, or a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

In another embodiment of the invention, the invention provides a methodfor producing 6-aminocaproic acid (6-ACA) by culturing a non-naturallyoccurring microbial organism having a 6-aminocaproic acid (6-ACA)pathway including a set of exogenous nucleic acids encoding 6-ACApathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto-aciddecarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of FIG. 26).In yet another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encoding6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of FIG. 26).In still yet another embodiment of the invention, the the non-naturallyoccurring microbial organism includes a set of exogenous nucleic acidsencoding 6-ACA pathway enzymes, where the set encodes a2-amino-7-oxosubarate amino acid decarboxylase; a2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanaloxidoreductase (see Example XXV; steps I/G/F of FIG. 26). In a furtheraspect of each of the above embodiments, the microbioal organism has a2-amino-7-oxosubarate pathway having a second set of exogenous nucleicacids encoding 2-amino-7-oxosubarate pathway enzymes expressed in asufficient amount to produce 2-amino-7-oxosubarate, the2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway, the microbial organismincluding at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA, the HMDApathway including a 6-aminocaproate kinase, an[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, a6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproicsemialdehyde oxidoreductase (aminating), a 6-aminocaproateN-acetyltransferase, a 6-acetamidohexanoate kinase, an[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase(aminating), a 6-acetamidohexanamine N-acetyltransferase, a6-acetamidohexanamine hydrolase (amide), a 6-acetamidohexanoate CoAtransferase, a 6-acetamidohexanoate CoA ligase, a6-acetamidohexanoyl-CoA oxidoreductase, a[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase, a[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase, a6-aminocaproate CoA transferase and a 6-aminocaproate CoA ligase (seeExamples XX and XXI; steps A-N of FIG. 13).

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway, the microbial organismincluding at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA. In one aspectthe HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOPoxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (seeExamples XX and XXI; steps A/B/C of FIG. 13). In another aspect of theinvention, the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOPacyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a6-aminocaproic semialdehyde oxidoreductase (aminating) or a6-aminocaproic acid semialdehyde aminotransferase (see Examples XX andXXI; steps A/L/N/C of FIG. 13). In another aspect of the invention, theHMDA pathway includes a 6-aminocaproate CoA transferase or a6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a6-aminocaproic semialdehyde oxidoreductase (aminating) or a6-aminocaproic acid semialdehyde aminotransferase (see Examples XX andXXI; steps M/N/C of FIG. 13). In another aspect of the invention, theHMDA pathway includes a 6-aminocaproate N-acetyltransferase; a6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a6-acetamidohexanal aminotransferase or a 6-acetamidohexanaloxidoreductase (aminating); and a 6-acetamidohexanamineN-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (seeExamples XX and XXI; steps D/E/F/G/H of FIG. 13). In another aspect ofthe invention, the HMDA pathway includes a 6-aminocaproateN-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoAoxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of FIG. 13).In another aspect of the invention, the HMDA pathway includes a6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a6-acetamidohexanal oxidoreductase (aminating); and a6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanaminehydrolase (amide) (see Examples XX and XXI; steps D/E/K/J/G of FIG. 13).

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an3-oxo-6-aminopimeloyl-CoA oxidoreductase, a3-hydroxy-6-aminopimeloyl-CoA dehydratase, a6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoAreductase (aldehyde forming), a 2-amino-7-oxoheptanoateaminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, ora homolysine decarboxylase (see Examples XXIV and XXVI; steps A-H ofFIG. 20). In another aspect of the invention, the non-naturallyoccurring microbial organism includes a set of exogenous nucleic acidsencoding HMDA pathway enzymes, wherein the set encodes a glutamyl-CoAtransferase or ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoAoxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferaseor aminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a3-oxo-1-carboxyheptanal aminating oxidoreductase, a3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase, a 3-oxopimelate kinase, a5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming),a 3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelateligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ahomolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase(aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV andXXVI; FIG. 21).

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ora homolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/C/D/E/R/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 7-aminotransferase ora 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminatingoxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a5-oxopimeloyl-CoA reductase (aldehyde forming), a3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoAtransferase or 3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase(aldehyde forming); a 3-oxo-1-carboxyheptanal 7-aminotransferase or3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ora homolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/C/AB/Z/R/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase ora 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelatekinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of FIG. 21). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a5-oxopimeloyl-CoA reductase (aldehyde forming), a3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase,a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of FIG. 21). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate CoA transferase or a3-oxopimelate CoA ligase; a 5-oxopimeloyl-CoA reductase (aldehydeforming); a 3-oxo-1-carboxyheptanal 3-aminotransferase or a3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B//J/O/P/Q/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or a 3-oxopimelate aminating oxidoreductase; a3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonatereductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase (see ExamplesXXIV and XXVI; steps A/B/J/M/N/P/Q/S of FIG. 21). In another aspect ofthe invention, the non-naturally occurring microbial organism includes aset of exogenous nucleic acids encoding HMDA pathway enzymes, whereinthe set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonatereductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoAligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, ora homolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/J/K/L/P/Q/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelateCoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase;and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase,a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of FIG. 21). In anotheraspect of the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding HMDA pathway enzymes,wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoAligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoateaminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or ahomolysine decarboxylase (see Examples XXIV and XXVI; stepsA/B/J/K/L/Z/R/S of FIG. 21). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelateCoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoateaminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonatereductase, a 3-amino-7-oxoheptanoate 7-aminotransferase, a3-amino-7-oxoheptanoate aminating oxidoreductase, a3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase(see Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S of FIG. 21). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate7-aminotransferase or a 3-amino-7-oxoheptanoate aminatingoxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase (see ExamplesXXIV and XXVI; steps A/B/J/T/W/Q/S of FIG. 21). In another aspect of theinvention, the non-naturally occurring microbial organism includes a setof exogenous nucleic acids encoding HMDA pathway enzymes, wherein theset encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelatereductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelatekinase, a 6-aminopimeloylphosphonate reductase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase (see ExamplesXXIV and XXVI; steps A/B/J/T/U/X/Q/S of FIG. 21). In another aspect ofthe invention, the non-naturally occurring microbial organism includes aset of exogenous nucleic acids encoding HMDA pathway enzymes, whereinthe set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoAligase; a 3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelatekinase; a 6-aminopimeloylphosphonate reductase; a2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase.

In yet another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a HMDA pathway including at least oneexogenous nucleic acid encoding a HMDA pathway enzyme expressed in asufficient amount to produce HMDA, the HMDA pathway including aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoAtransferase, 2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase(aldehyde forming), a 2-amino-7-oxoheptanoate 7-aminotransferase,2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysinedecarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of FIG.21). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a glutaryl-CoAbeta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoAtransferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelateaminotransferase or a 3-oxopimelate aminating oxidoreductase; a3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehydeforming); a 2-amino-7-oxoheptanoate 7-aminotransferase or2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase.

The invention additionally provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a hexamethylenediamine (HMDA) pathwayincluding at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA, the HMDApathway including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoateaminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminatingoxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoatedecarboxylase, a 6-aminohexanal aminotransferase or 6-aminohexanalaminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of FIG.22). In another aspect of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoateaminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;and a homolysine decarboxylase. In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes a2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoatedecarboxylase; and a 6-aminohexanal aminotransferase or a 6-aminohexanalaminating oxidoreductase.

The invention additionally provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a hexamethylenediamine (HMDA) pathwayincluding at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA, the HMDApathway including a 6-aminocaproate reductase, a 6-aminocaproicsemialdehyde aminotransferase, a 6-aminocaproic semialdehydeoxidoreductase (aminating), 6-aminocaproate N-acetyltransferase, a6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase,6-acetamidohexanal oxidoreductase (aminating), 6-acetamidohexanamineN-acetyltransferase or acetamidohexanamine hydrolase (amide) (seeExample XXVII; steps 0/C or D/P/G/H of FIG. 24). In another aspect ofthe invention, the non-naturally occurring microbial organism includes aset of exogenous nucleic acids encoding HMDA pathway enzymes, whereinthe set encodes a 6-aminocaproate reductase; and a 6-aminocaproicsemialdehyde aminotransferase or a 6-aminocaproic semialdehydeoxidoreductase (aminating). In another aspect of the invention, thenon-naturally occurring microbial organism includes a set of exogenousnucleic acids encoding HMDA pathway enzymes, wherein the set encodes6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate reductase;6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase(aminating); and 6-acetamidohexanamine N-acetyltransferase or6-acetamidohexanamine hydrolase (amide).

The invention additionally provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a hexamethylenediamine (HMDA) pathwayincluding at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA, the HMDApathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a2-amino-7-oxoheptanoate decarboxylase, a 6-aminohexanal aminatingoxidoreductase, a 6-aminohexanal aminotransferase, a2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, a 2-oxo-7-aminoheptanoate decarboxylase, ahomolysine decarboxylase, a 2-amino-7-oxosubarate amino aciddecarboxylase, a 2-oxo-7-aminoheptanoate aminating oxidoreductase, a2-oxo-7-aminoheptanoate aminotransferase, a 2-amino-7-oxosubarateaminating oxidoreductase, a 2-amino-7-oxosubarate aminotransferase or a2,7-diaminosubarate decarboxylase (see Examples XXIV and XXVI; StepsA/B/C/G/H/I/J/K/L/M of FIG. 26). In a further aspect, the microbioalorganism has a 2-amino-7-oxosubarate pathway having at least oneexogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzymeexpressed in a sufficient amount to produce 2-amino-7-oxosubarate, the2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase, a2-amino-5-hydroxy-7-oxosubarate dehydratase, or a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

In another embodiment, the invention provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a hexamethylenediamine (HMDA) pathwayincluding a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a 2-amino-7-oxosubarate aminatingoxidoreductase or 2-amino-7-oxosubarate aminotransferase; a2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (seeExamples XXIV and XXVI; steps K/L/H of FIG. 26). In another embodimentof the invention, the non-naturally occurring microbial organismincludes a set of exogenous nucleic acids encoding HMDA pathway enzymes,wherein the set encodes a 2-amino-7-oxosubarate amino aciddecarboxylase; a 2-oxo-7-aminoheptanoate aminating oxidoreductase or a2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase(see Examples XXIV and XXVI; steps I/J/H of FIG. 26). In anotherembodiment of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding HMDA pathwayenzymes, wherein the set encodes a 2-amino-7-oxosubarate amino aciddecarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a6-aminohexanal aminating oxidoreductase or a 6-aminohexanalaminotransferase (see Examples XXIV and XXVI; steps I/G/C of FIG. 26).In another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a6-aminohexanal aminating oxidoreductase or a 6-aminohexanalaminotransferase (see Examples XXIV and XXVI; steps A/B/C of FIG. 26).In another embodiment of the invention, the non-naturally occurringmicrobial organism includes a set of exogenous nucleic acids encodingHMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate aminatingoxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and ahomolysine decarboxylase (see Examples XXIV and XXVI; steps A/M/H ofFIG. 26). In a further aspect of each of the above embodiments, themicrobioal organism has a 2-amino-7-oxosubarate pathway having a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway including a2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; stepsA/B/C of FIG. 27).

The invention additionally provides a method for producinghexamethylenediamine (HMDA) by culturing a non-naturally occurringmicrobial organism having a levulinic acid (LA) pathway including atleast one exogenous nucleic acid encoding a LA pathway enzyme expressedin a sufficient amount to produce LA, the LA pathway including a3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA/acyl-CoA transferase, a3-oxoadipyl-CoA synthase, a 3-oxoadipyl-CoA hydrolase, or a 3-oxoadipatedecarboxylase (see Example XXIX; steps A/E/F/G/AA of FIG. 25). Inanother aspect of the invention, the non-naturally occurring microbialorganism includes a set of exogenous nucleic acids encoding LA pathwayenzymes, wherein the set encodes a 3-oxoadipyl-CoA thiolase; a3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, or a3-oxoadipyl-CoA hydrolase; and a 3-oxoadipate decarboxylase.

The invention further provides methods of producing non-naturallymicrobial organisms having increased production of adipate, 6-ACA and/orHMDA by disruption of one or more genes to confer increased productionof adiate, 6-ACA and/or HMDA. Such gene disruptions include thoseexemplified herein in Example XXX and Tables 14-16.

The invention additionally provides a method for producing adipate,6-ACA and/or HMDA that includes culturing a non-naturally occurringmicrobial organism that includes one or more gene disruptions thatconfer increased production of adiapte, 6-ACA and/or HMDA. Thedisruptions can occur in genes encoding an enzyme obligatory to couplingadipate, 6-ACA and/or HMDA production to growth of the microorganismwhen the gene disruption reduces an activity of the enzyme, such thatthe disruptions confer stable growth-coupled production of adipate,6-ACA and/or HMDA onto the non-naturally microbial organism.

In some embodiments, the gene disruption can include a complete genedeletion. Methods for gene disruption are well known to those skilled inthe art and are described herein (see Example XXX). In some embodimentsother methods to disrupt a gene include, for example, frameshifting byomission, addition of oligonucleotides or by mutations that render thegene inoperable. One skilled in the art will recognize the advantages ofgene deletions, however, because of the stability it can confer to thenon-naturally occurring organism from reverting to a phenotypeexpressing the previously disrupted gene. In particular, the genedisruptions are selected from the gene sets that described in Tables14-16.

Suitable purification and/or assays to test for the production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidcan be performed using well known methods. Suitable replicates such astriplicate cultures can be grown for each engineered strain to betested. For example, product and byproduct formation in the engineeredproduction host can be monitored. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) orother suitable analytical methods using routine procedures well known inthe art. The release of product in the fermentation broth can also betested with the culture supernatant. Byproducts and residual glucose canbe quantified by HPLC using, for example, a refractive index detectorfor glucose and alcohols, and a UV detector for organic acids (Lin etal., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay anddetection methods well known in the art. The individual enzymeactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art.

The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid can be separated from other components in the culture using avariety of methods well known in the art. Such separation methodsinclude, for example, extraction procedures as well as methods thatinclude continuous liquid-liquid extraction, pervaporation, membranefiltration, membrane separation, reverse osmosis, electrodialysis,distillation, crystallization, centrifugation, extractive filtration,ion exchange chromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid producers can be cultured for thebiosynthetic production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid.

For the production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid, the recombinant strains arecultured in a medium with carbon source and other essential nutrients.It is sometimes desirable and can be highly desirable to maintainanaerobic conditions in the fermenter to reduce the cost of the overallprocess. Such conditions can be obtained, for example, by first spargingthe medium with nitrogen and then sealing the flasks with a septum andcrimp-cap. For strains where growth is not observed anaerobically,microaerobic or substantially anaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in United State publication 2009/0047719 (Ser. No.11/891,602), filed Aug. 10, 2007. Fermentations can be performed in abatch, fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid.

In addition to renewable feedstocks such as those exemplified above, the6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidmicrobial organisms of the invention also can be modified for growth onsyngas as its source of carbon. In this specific embodiment, one or moreproteins or enzymes are expressed in the 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid producing organismsto provide a metabolic pathway for utilization of syngas or othergaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂ +nADP+nPi→CH3COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: cobalamide corrinoid/iron-sulfurprotein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoAsynthase, acetyl-CoA synthase disulfide reductase and hydrogenase, andthese enzymes can also be referred to asmethyltetrahydrofolate:corrinoid protein methyltransferase (for example,AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein(for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxidedehydrogenase and nickel-protein assembly protein (for example, CooC).Following the teachings and guidance provided herein for introducing asufficient number of encoding nucleic acids to generate a 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupledwith carbon monoxide dehydrogenase and/or hydrogenase activities canalso be used for the conversion of CO, CO₂ and/or H₂ to acetyl-CoA andother products such as acetate. Organisms capable of fixing carbon viathe reductive TCA pathway can utilize one or more of the followingenzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitratedehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase,fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase,carbon monoxide dehydrogenase, and hydrogenase. Specifically, thereducing equivalents extracted from CO and/or H₂ by carbon monoxidedehydrogenase and hydrogenase are utilized to fix CO₂ via the reductiveTCA cycle into acetyl-CoA or acetate. Acetate can be converted toacetyl-CoA by enzymes such as acetyl-CoA transferase, acetatekinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA canbe converted to the p-toluate, terepathalate, or(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors,glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, bypyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis.Following the teachings and guidance provided herein for introducing asufficient number of encoding nucleic acids to generate a p-toluate,terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway,those skilled in the art will understand that the same engineeringdesign also can be performed with respect to introducing at least thenucleic acids encoding the reductive TCA pathway enzymes or proteinsabsent in the host organism. Therefore, introduction of one or moreencoding nucleic acids into the microbial organisms of the inventionsuch that the modified organism contains the complete reductive TCApathway will confer syngas utilization ability.

Given the teachings and guidance provided herein, those skilled in theart will understand that a non-naturally occurring microbial organismcan be produced that secretes the biosynthesized compounds of theinvention when grown on a carbon source such as a carbohydrate. Suchcompounds include, for example, 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid and any of the intermediatemetabolites in the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway. All that is required isto engineer in one or more of the required enzyme activities to achievebiosynthesis of the desired compound or intermediate including, forexample, inclusion of some or all of the 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid biosyntheticpathways. Accordingly, the invention provides a non-naturally occurringmicrobial organism that produces and/or secretes 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid when grown on acarbohydrate and produces and/or secretes any of the intermediatemetabolites shown in the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid pathway when grown on acarbohydrate. For example, an adipate producing microbial organisms caninitiate synthesis from an intermediate, for example, 3-oxoadipyl-CoA,3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA, or adipyl-CoA (see FIG.2), as desired. In addition, an adipate producing microbial organism caninitiate synthesis from an intermediate, for example, 3-oxoadipyl-CoA,3-oxoadipate, 3-hydroxyadipate, or hexa-2-enedioate (see FIG. 3). The6-aminocaproic acid producing microbial organism of the invention caninitiate synthesis from an intermediate, for example, adipatesemialdehyde (see FIG. 8). The caprolactam producing microbial organismof the invention can initiate synthesis from an intermediate, forexample, adipate semialdehyde or 6-aminocaproic acid (see FIG. 8), asdesired.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid pathway enzymein sufficient amounts to produce 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid. It is understood that themicrobial organisms of the invention are cultured under conditionssufficient to produce 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid. Following the teachings andguidance provided herein, the non-naturally occurring microbialorganisms of the invention can achieve biosynthesis of 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid resulting inintracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid is between about3-150 mM, particularly between about 5-125 mM and more particularlybetween about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, ormore. Intracellular concentrations between and above each of theseexemplary ranges also can be achieved from the non-naturally occurringmicrobial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicconditions, the 6-aminocaproic acid, caprolactam, hexamethylenediamineor levulinic acid producers can synthesize 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid at intracellularconcentrations of 5-10 mM or more as well as all other concentrationsexemplified herein. It is understood that, even though the abovedescription refers to intracellular concentrations, 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid producing microbialorganisms can produce 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid intracellularly and/or secretethe product into the culture medium.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamineor levulinic acid includes anaerobic culture or fermentation conditions.In certain embodiments, the non-naturally occurring microbial organismsof the invention can be sustained, cultured or fermented under anaerobicor substantially anaerobic conditions. Briefly, anaerobic conditionsrefers to an environment devoid of oxygen. Substantially anaerobicconditions include, for example, a culture, batch fermentation orcontinuous fermentation such that the dissolved oxygen concentration inthe medium remains between 0 and 10% of saturation. Substantiallyanaerobic conditions also includes growing or resting cells in liquidmedium or on solid agar inside a sealed chamber maintained with anatmosphere of less than 1% oxygen. The percent of oxygen can bemaintained by, for example, sparging the culture with an N₂/CO₂ mixtureor other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid. Generally,and as with non-continuous culture procedures, the continuous and/ornear-continuous production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid will include culturing anon-naturally occurring 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid producing organism of theinvention in sufficient nutrients and medium to sustain and/or nearlysustain growth in an exponential phase. Continuous culture under suchconditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days ormore. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5or more weeks and up to several months. Alternatively, organisms of theinvention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid can be utilized in, for example,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation. Examples of batch and continuous fermentation procedures arewell known in the art.

In addition to the above fermentation procedures using the6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidproducers of the invention for continuous production of substantialquantities of 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid, the 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid producers also can be, forexample, simultaneously subjected to chemical synthesis procedures toconvert the product to other compounds or the product can be separatedfrom the fermentation culture and sequentially subjected to chemicalconversion to convert the product to other compounds, if desired. Asdescribed herein, an intermediate in the adipate pathway utilizing3-oxoadipate, hexa-2-enedioate, can be converted to adipate, forexample, by chemical hydrogenation over a platinum catalyst (see ExampleIII).

As described herein, exemplary growth conditions for achievingbiosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamineor levulinic acid includes the addition of an osmoprotectant to theculturing conditions. In certain embodiments, the non-naturallyoccurring microbial organisms of the invention can be sustained,cultured or fermented as described above in the presence of anosmoprotectant. Briefly, an osmoprotectant means a compound that acts asan osmolyte and helps a microbial organism as described herein surviveosmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.For example, as described in Example XXII, Escherichia coli in thepresence of varying amounts of 6-aminocaproic acid is suitably grown inthe presence of 2 mM glycine betaine. The amount of osmoprotectant inthe culturing conditions can be, for example, no more than about 0.1 mM,no more than about 0.5 mM, no more than about 1.0 mM, no more than about1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no morethan about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM,no more than about 10 mM, no more than about 50 mM, no more than about100 mM or no more than about 500 mM.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework, Burgard et al., Biotechnol. Bioeng. 84:647-657(2003). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

The concept of growth-coupled biochemical production can be visualizedin the context of the biochemical production envelopes of a typicalmetabolic network calculated using an in silico model. These limits areobtained by fixing the uptake rate(s) of the limiting substrate(s) totheir experimentally measured value(s) and calculating the maximum andminimum rates of biochemical production at each attainable level ofgrowth. Although exceptions exist, typically the production of a desiredbiochemical is in direct competition with biomass formation forintracellular resources. Thus, enhanced rates of biochemical productionwill necessarily result in sub-maximal growth rates. The knockoutssuggested by OptKnock are designed to restrict the allowable solutionboundaries forcing a change in metabolic behavior from the wild-typestrain. Although the actual solution boundaries for a given strain willexpand or contract as the substrate uptake rate(s) increase or decrease,each experimental point should lie within its calculated solutionboundary. Plots such as these allow one to visualize how close strainsare to their performance limits or, in other words, how much room isavailable for improvement. The OptKnock framework has already been ableto identify promising gene deletion strategies for biochemicaloverproduction, (Burgard et al., Biotechnol Bioeng, 84(6):647-657(2003); Pharkya et al., Biotechnol Bioeng, 84(7):887-899 (2003)) andestablishes a systematic framework that will naturally encompass futureimprovements in metabolic and regulatory modeling frameworks.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethod referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patentapplication serial No. 2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.Analysis methods such as convex analysis, linear programming and thecalculation of extreme pathways as described, for example, in Schillinget al., J. Theor. Biol. 203:229-248 (2000); Schilling et al., Biotech.Bioeng. 71:286-306 (2000) and Schilling et al., Biotech. Prog.15:288-295 (1999), can be used to determine such phenotypiccapabilities.

As described above, one constraints-based method used in thecomputational programs applicable to the invention is flux balanceanalysis. Flux balance analysis is based on flux balancing in a steadystate condition and can be performed as described in, for example, Varmaand Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approacheshave been applied to reaction networks to simulate or predict systemicproperties of, for example, adipocyte metabolism as described in Felland Small, J. Biochem. 138:781-786 (1986), acetate secretion from E.coli under ATP maximization conditions as described in Majewski andDomach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeastas described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996).Additionally, this approach can be used to predict or simulate thegrowth of S. cerevisiae on a variety of single-carbon sources as well asthe metabolism of H. influenzae as described in Edwards and Palsson,Proc. Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J. Bio.Chem. 274:17410-17416 (1999) and Edwards et al., Nature Biotech.19:125-130 (2001).

Once the solution space has been defined, it can be analyzed todetermine possible solutions under various conditions. Thiscomputational approach is consistent with biological realities becausebiological systems are flexible and can reach the same result in manydifferent ways. Biological systems are designed through evolutionarymechanisms that have been restricted by fundamental constraints that allliving systems must face. Therefore, constraints-based modeling strategyembraces these general realities. Further, the ability to continuouslyimpose further restrictions on a network model via the tightening ofconstraints results in a reduction in the size of the solution space,thereby enhancing the precision with which physiological performance orphenotype can be predicted.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The ability of a cell or organism to obligatory couple growth to theproduction of a biochemical product can be illustrated in the context ofthe biochemical production limits of a typical metabolic networkcalculated using an in silico model. These limits are obtained by fixingthe uptake rate(s) of the limiting substrate(s) to their experimentallymeasured value(s) and calculating the maximum and minimum rates ofbiochemical production at each attainable level of growth. Theproduction of a desired biochemical generally is in direct competitionwith biomass formation for intracellular resources. Under thesecircumstances, enhanced rates of biochemical production will necessarilyresult in sub-maximal growth rates. The knockouts suggested by the abovemetabolic modeling and simulation programs such as OptKnock are designedto restrict the allowable solution boundaries forcing a change inmetabolic behavior from the wild-type strain. Although the actualsolution boundaries for a given strain will expand or contract as thesubstrate uptake rate(s) increase or decrease, each experimental pointwill lie within its calculated solution boundary. Plots such as theseallow accurate predictions of how close the designed strains are totheir performance limits which also indicates how much room is availablefor improvement.

The OptKnock mathematical framework is exemplified herein forpinpointing gene deletions leading to growth-coupled biochemicalproduction (see Example XXX). The procedure builds upon constraint-basedmetabolic modeling which narrows the range of possible phenotypes that acellular system can display through the successive imposition ofgoverning physico-chemical constraints, Price et al., Nat Rev Microbiol,2: 886-97 (2004). As described above, constraint-based models andsimulations are well known in the art and generally invoke theoptimization of a particular cellular objective, subject to networkstoichiometry, to suggest a likely flux distribution.

Briefly, the maximization of a cellular objective quantified as anaggregate reaction flux for a steady state metabolic network comprisinga set N={1, . . . , N} of metabolites and a set M={1, . . . , M} ofmetabolic reactions is expressed mathematically as follows:

  maximize  v_(cellular  objective)$\mspace{20mu} {{{{subject}\mspace{14mu} {to}\underset{j = 1}{\overset{M}{\;\sum}}\; S_{ij}v_{j}} = 0},{\forall{i \in N}}}$v_(substrate) = v_(substrate_uptake)mmol/gDW ⋅ hr  ∀i ∈ {limiting  substrate(s)}  v_(atp) ≥ v_(atp_main)mmol/gDW ⋅ hr  v_(j) ≥ 0, ∀j ∈ {irrev. reactions}

where S_(ij) is the stoichiometric coefficient of metabolite i inreaction j, v_(j) is the flux of reaction j, v_(substrate_uptake)represents the assumed or measured uptake rate(s) of the limitingsubstrate(s), and v_(atp_main) is the non-growth associated ATPmaintenance requirement. The vector v includes both internal andexternal fluxes. In this study, the cellular objective is often assumedto be a drain of biosynthetic precursors in the ratios required forbiomass formation, Neidhardt, F. C. et al., 2nd ed. 1996, Washington,D.C.: ASM Press. 2 v. (xx, 2822, lxxvi). The fluxes are generallyreported per 1 gDW·hr (gram of dry weight times hour) such that biomassformation is expressed as g biomass produced/gDW·hr or 1/hr.

The modeling of gene deletions, and thus reaction elimination, firstemploys the incorporation of binary variables into the constraint-basedapproach framework, Burgard et al., Biotechnol Bioeng, 74: 364-375(2001), Burgard et al., Biotechnol Prog, 17: 791-797 (2001). Thesebinary variables,

$y_{j} = \left\{ {\begin{matrix}{1,{{if}\mspace{14mu} {reaction}\mspace{14mu} {flux}\mspace{14mu} v_{j}\mspace{14mu} {is}\mspace{14mu} {active}}} \\{0,{{if}\mspace{14mu} {reaction}\mspace{14mu} {flux}\mspace{14mu} v_{j}\mspace{14mu} {is}\mspace{14mu} {not}\mspace{14mu} {active}}}\end{matrix},{\forall{j \in M}}} \right.$

assume a value of 1 if reaction j is active and a value of 0 if it isinactive. The following constraint,

v _(j) ^(min) ·y _(j) ≤v _(j) ≤v _(j) ^(max) ·y _(j) ,∀j∈M

ensures that reaction flux v_(j) is set to zero only if variable y_(j)is equal to zero. Alternatively, when y_(j) is equal to one, v_(j) isfree to assume any value between a lower v_(j) ^(min) and an upper v_(j)^(max) bound. Here, v_(j) ^(min) and v_(j) ^(max) are identified byminimizing and maximizing, respectively, every reaction flux subject tothe network constraints described above, Mahadevan et al., Metab Eng, 5:264-76 (2003).

Optimal gene/reaction knockouts are identified by solving a bileveloptimization problem that chooses the set of active reactions (y_(j)=1)such that an optimal growth solution for the resulting networkoverproduces the chemical of interest. Schematically, this bileveloptimization problem is illustrated in FIG. 2. Mathematically, thisbilevel optimization problem is expressed as the following bilevelmixed-integer optimization problem:

$\mspace{79mu} {\underset{y_{j}}{maximize}\mspace{14mu} v_{chemical}\mspace{14mu} ({OptKnock})}$$\begin{pmatrix}\underset{y_{j}}{{subject}\mspace{14mu} {to}} & {maximize} & v_{biomass} & \; \\\; & {{subject}\mspace{14mu} {to}} & {{\sum\limits_{j = 1}^{M}\; {S_{ij}v_{j}}} = 0} & {\forall{i \in N}} \\\; & \; & {v_{substrate} = v_{s{ubstrate}\_ {uptake}}} & {\forall{i \in \left\{ {{limiting}\mspace{14mu} {{substrate}(s)}} \right\}}} \\\; & \; & {v_{atp} \geq v_{{atp}\_ {main}}} & \; \\\; & \; & {v_{biomass} \geq v_{biomass}^{target}} & \;\end{pmatrix}$   v_(j)^(min).y_(j) ≤ v_(j) ≤ v_(j)^(max).y_(j), ∀j ∈ M$\mspace{20mu} {{\sum\limits_{j = M^{forward}}\; \left( {1 - y_{j}} \right)} = K}$  y_(j) ∈ {0, 1}, ∀j ∈ M

where v_(chemical) is the production of the desired target product, forexample adipate, 6-ACA and/or HMDA, or other biochemical product, and Kis the number of allowable knockouts. Note that setting K equal to zeroreturns the maximum biomass solution of the complete network, whilesetting K equal to one identifies the single gene/reaction knockout(y_(j)=0) such that the resulting network involves the maximumoverproduction given its maximum biomass yield. The final constraintensures that the resulting network meets a minimum biomass yield.Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), provide a moredetailed description of the model formulation and solution procedure.Problems containing hundreds of binary variables can be solved in theorder of minutes to hours using CPLEX 8.0, GAMS: The Solver Manuals.2003: GAMS Development Corporation, accessed via the GAMS, Brooke etal., GAMS Development Corporation (1998), modeling environment on an IBMRS6000-270 workstation. The OptKnock framework has already been able toidentify promising gene deletion strategies for biochemicaloverproduction, Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003),Pharkya et al., Biotechnol Bioeng, 84: 887-899 (2003), and establishes asystematic framework that will naturally encompass future improvementsin metabolic and regulatory modeling frameworks.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

Given the teachings and guidance provided herein, those skilled in theart will understand that to disrupt an enzymatic reaction the catalyticactivity of the one or more enzymes involved in the reaction is to bedisrupted. Disruption can occur by a variety of means including, forexample, deletion of an encoding gene or incorporation of a geneticalteration in one or more of the encoding gene sequences. The encodinggenes targeted for disruption can be one, some, or all of the genesencoding enzymes involved in the catalytic activity. For example, wherea single enzyme is involved in a targeted catalytic activity disruptioncan occur by a genetic alteration that reduces or destroys the catalyticactivity of the encoded gene product. Similarly, where the single enzymeis multimeric, including heteromeric, disruption can occur by a geneticalteration that reduces or destroys the function of one or all subunitsof the encoded gene products. Destruction of activity can beaccomplished by loss of the binding activity of one or more subunits inorder to form an active complex, by destruction of the catalytic subunitof the multimeric complex or by both. Other functions of multimericprotein association and activity also can be targeted in order todisrupt a metabolic reaction of the invention. Such other functions arewell known to those skilled in the art. Further, some or all of thefunctions of a single polypeptide or multimeric complex can be disruptedaccording to the invention in order to reduce or abolish the catalyticactivity of one or more enzymes involved in a reaction or metabolicmodification of the invention. Similarly, some or all of enzymesinvolved in a reaction or metabolic modification of the invention can bedisrupted so long as the targeted reaction is reduced or eliminated.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the growth-coupled product production.Exemplary disruptions to confer increased production of adipate, 6-ACAand/or HMDA are described in Example XXX and Tables 14-16.

Employing the methods exemplified above, the methods of the inventionallow the construction of cells and organisms that increase productionof a desired product, for example, by coupling the production of adesired product to growth of the cell or organism engineered to harborthe identified genetic alterations. As disclosed herein, metabolicalterations have been identified that couple the production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidto growth of the organism. Microbial organism strains constructed withthe identified metabolic alterations produce elevated levels, relativeto the absence of the metabolic alterations, of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid during theexponential growth phase. These strains can be beneficially used for thecommercial production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid in continuous fermentationprocess without being subjected to the negative selective pressuresdescribed previously. Although exemplified herein as metabolicalterations, in particular one or more gene disruptions, that confergrowth coupled production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid, it is understood that any genedisruption that increases the production of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid can be introducedinto a host microbial organism, as desired.

Therefore, the methods of the invention provide a set of metabolicmodifications that are identified by an in silico method such asOptKnock. The set of metabolic modifications can include functionaldisruption of one or more metabolic reactions including, for example,disruption by gene deletion. For 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid production, metabolicmodifications can be selected from the set of metabolic modificationslisted in Tables 14-16 (see Example XXX).

Also provided is a method of producing a non-naturally occurringmicrobial organisms having stable growth-coupled production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid. The method can include identifying in silico a set of metabolicmodifications that increase production of 6-aminocaproic acid,caprolactam, hexamethylenediamine or levulinic acid, for example,increase production during exponential growth; genetically modifying anorganism to contain the set of metabolic modifications that increaseproduction of 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid, and culturing the genetically modified organism. Ifdesired, culturing can include adaptively evolving the geneticallymodified organism under conditions requiring production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinicacid. The methods of the invention are applicable to bacterium, yeastand fungus as well as a variety of other cells and microorganism, asdisclosed herein.

Thus, the invention provides a non-naturally occurring microbialorganism comprising one or more gene disruptions that confer increasedproduction of 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid. In one embodiment, the one or more gene disruptionsconfer growth-coupled production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid, and can, for example, conferstable growth-coupled production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid. In another embodiment, the oneor more gene disruptions can confer obligatory coupling of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidproduction to growth of the microbial organism. Such one or more genedisruptions reduce the activity of the respective one or more encodedenzymes.

The non-naturally occurring microbial organism can have one or more genedisruptions included in a metabolic modification listed in Tables 14-16.As disclosed herein, the one or more gene disruptions can be a deletion.Such non-naturally occurring microbial organisms of the inventioninclude bacteria, yeast, fungus, or any of a variety of othermicroorganisms applicable to fermentation processes, as disclosedherein.

Thus, the invention provides a non-naturally occurring microbialorganism, comprising one or more gene disruptions, where the one or moregene disruptions occur in genes encoding proteins or enzymes where theone or more gene disruptions confer increased production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidin the organism. The production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid can be growth-coupled or notgrowth-coupled. In a particular embodiment, the production of6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acidcan be obligatorily coupled to growth of the organism, as disclosedherein.

The invention provides non naturally occurring microbial organismshaving genetic alterations such as gene disruptions that increaseproduction of 6-aminocaproic acid, caprolactam, hexamethylenediamine orlevulinic acid, for example, growth-coupled production of 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid. Productproduction can be, for example, obligatorily linked to the exponentialgrowth phase of the microorganism by genetically altering the metabolicpathways of the cell, as disclosed herein. The genetic alterations canincrease the production of the desired product or even make the desiredproduct an obligatory product during the growth phase. Sets of metabolicalterations or transformations that result in increased production andelevated levels of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid biosynthesis are exemplified inTables 14-16 (see Example XXX). Each alteration within a set correspondsto the requisite metabolic reaction that should be functionallydisrupted. Functional disruption of all reactions within each set canresult in the increased production of 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid by the engineered strain duringthe growth phase. The corresponding reactions to the referencedalterations can be found in Tables 14-16 (see Example XXX), and the geneor genes that encode enzymes or proteins that carry out the reactionsare set forth in Tables 14-16.

For example, for each strain exemplified in Tables 14-16, the metabolicalterations that can be generated for 6-aminocaproic acid, caprolactam,hexamethylenediamine or levulinic acid production are shown in each row.These alterations include the functional disruption of the reactionsshown in Tables 14-16. Each of these non-naturally occurring alterationsresult in increased production and an enhanced level of 6-aminocaproicacid, caprolactam, hexamethylenediamine or levulinic acid production,for example, during the exponential growth phase of the microbialorganism, compared to a strain that does not contain such metabolicalterations, under appropriate culture conditions. Appropriateconditions include, for example, those disclosed herein, includingconditions such as particular carbon sources or reactant availabilitiesand/or adaptive evolution.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Reverse Adipate Degradation Pathway

This example describes an exemplary adipate synthesis pathway via areverse adipate degradation pathway.

Organisms such as Penicillium chrysogenum have the ability to naturallydegrade adipate (Thykaer et al., Metab. Eng. 4:151-158. (2002)). Themechanism is similar to the oxidation of fatty acids (see FIG. 1). Thefirst step in adipate degradation is an ATP-dependent reaction thatactivates adipate with CoA. The second reaction is catalyzed by adehydrogenase that forms 5-carboxy-2-pentenoyl-CoA from adipyl-CoA.During peroxisomal adipate degradation, the dehydrogenase enzymecontains FAD, which accepts the electrons and then transfers themdirectly to oxygen. A catalase enzyme dissipates the H₂O₂ formed by thereduction of oxygen. In mitochondrial fatty acid oxidation, the FAD fromthe dehydrogenase transfers electrons directly to the electron transportchain. A multi-functional fatty acid oxidation protein in eukaryotessuch as S. cerevisiae and P. chrysogenum carries out the followinghydratase and dehydrogenase steps. The final step is an acyl transferasethat splits 3-oxoadipyl CoA into acetyl-CoA and succinyl-CoA.

A highly efficient pathway for the production of adipate is achievedthrough genetically altering a microorganism such that similar enzymaticreactions are employed for adipate synthesis from succinyl-CoA andacetyl-CoA (see FIG. 2). Successful implementation of this entailsexpressing the appropriate genes, tailoring their expression, andaltering culture conditions so that high acetyl-CoA, succinyl-CoA,and/or redox (for example, NADH/NAD+) ratios will drive the metabolicflux through this pathway in the direction of adipate synthesis ratherthan degradation. Strong parallels to butyrate formation in Clostridia(Kanehisa and Goto, Nucl. Acids Res. 28:27-30 (2000)) support that eachstep in the adipate synthesis pathway is thermodynamically feasible withreaction directionality governed by the concentrations of theparticipating metabolites. The final step, which forms adipate fromadipyl-CoA, can take place either via a synthetase,phosphotransadipylase/kinase, transferase, or hydrolase mechanism.

The maximum theoretical yields of adipate using this pathway werecalculated both in the presence and absence of an external electronacceptor such as oxygen. These calculations show that the pathway canefficiently transform glucose into adipate and CO₂ under anaerobicconditions with a 92% molar yield (Table 1). The production of adipateusing this pathway does not require the uptake of oxygen as NAD+ can beregenerated in the two hydrogenase steps that form 3-hydroxyadipyl-CoAand adipyl-CoA (see FIG. 2). Further, the pathway is favorableenergetically as up to 1.55 moles of ATP are formed per mole of glucoseconsumed at the maximum theoretical yield of adipate assuming either asynthetase, phosphotransadipylase/kinase, or transferase mechanism forthe final conversion step. The ATP yield can be further improved to 2.47moles of ATP produced per mole of glucose if phosphoenolpyruvatecarboxykinase (PPCK) is assumed to function in the ATP-generatingdirection towards oxaloacetate formation. Maximum ATP yield calculationswere then performed assuming that the adipyl-CoA to adipatetransformation is a hydrolysis step. This reduces the maximum ATP yieldsat maximum adipate production to 0.85 and 1.77 mole ATP per mole glucoseconsumed if PPCK is assumed irreversible and reversible, respectively.Nevertheless, these ATP yields are sufficient for cell growth,maintenance, and production.

TABLE 1 The maximum theoretical yields of adipate and the associated ATPyields per mole of glucose using the reverse degradation pathwayassuming the final step in the pathway is a synthetase,phosphotransadipylase/kinase, or transferase. Aerobic Anaerobic AdipateYield 0.92 0.92 Max ATP yield @ max adipate yield 1.55 1.55 Max ATPyield @ max adipate yield 2.47 2.47 PPCK assumed

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of adipate, one or moreexogenous DNA sequence(s) are expressed in a suitable hostmicroorganism. In addition, the microorganisms can have endogenousgene(s) functionally deleted. These modifications allow the productionof adipate using renewable feedstock.

Below is described a number of biochemically characterized candidategenes that encode enzymes that catalyze each step of the reverse adipatedegradation pathway in a production host. Although described using E.coli as a host organism to engineer the pathway, essentially anysuitable host organism can be used. Specifically listed are genes thatare native to E. coli as well as genes in other organisms that can beapplied to catalyze the appropriate transformations when properly clonedand expressed.

Referring to FIG. 2, step 1 involves succinyl CoA:acetyl CoA acyltransferase ((3-ketothiolase). The first step in the pathway combinesacetyl-CoA and succinyl-CoA to form 3-oxoadipyl-CoA. The gene productsencoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J.Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera etal., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), paaE inPseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol.188:117-125 (2007)), and paaI from E. coli (Nogales et al., Microbiol.153:357-365 (2007)) catalyze the conversion of 3-oxoadipyl-CoA intosuccinyl-CoA and acetyl-CoA during the degradation of aromatic compoundssuch as phenylacetate or styrene. Since β-ketothiolase enzymes catalyzereversible transformations, these enzymes can be employed for the firststep in adipate synthesis shown in FIG. 2. For example, the ketothiolasephaA from R. eutropha combines two molecules of acetyl-CoA to formacetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)).Similarly, a β-keto thiolase (bktB) has been reported to catalyze thecondensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA(Slater et al., J. Bacteriol. 180: 1979-1987 (1998)) in R. eutropha.Additional candidates are found in Burkholderia ambifaria AMMD. Theprotein sequences for the above-mentioned gene products are well knownin the art and can be accessed in the public databases such as GenBankusing the following GI numbers and/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida paaE 106636097 ABF82237.1Pseudomonas fluorescens Bamb_0447 115350501 YP_772340 Burkholderiaambifaria AMMD

These exemplary sequences can be used to identify homologue proteins inGenBank or other databases through sequence similarity searches (forexample, BLASTp). The resulting homologue proteins and theircorresponding gene sequences provide additional exogenous DNA sequencesfor transformation into E. coli or other suitable host microorganisms togenerate production hosts.

For example, orthologs of paaJ from Escherichia coli K12 can be foundusing the following GI numbers and/or GenBank identifiers:

GI# GenBank Accession # Organism 152970031 YP_001335140.1 Klebsiellapneumoniae 157371321 YP_001479310.1 Serratia proteamaculans 3253200AAC24332.1 Pseudomonas putida

Example orthologs of pcaF from Pseudomonas knackmussii can be foundusing the following GI numbers and/or GenBank identifiers:

GI# GenBank Accession # Organism 4530443 AAD22035.1 Streptomyces sp.2065 24982839 AAN67000.1 Pseudomonas putida 115589162 ABJ15177.1Pseudomonas aeruginosa

Additional native candidate genes for the ketothiolase step includeatoB, which can catalyze the reversible condensation of 2 acetyl-CoAmolecules (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)), andits homolog yqeF. Non-native gene candidates include phaA (Sato et al.,supra, 2007) and bktB (Slater et al., J. Bacteriol. 180:1979-1987(1998)) from R. eutropha, and the two ketothiolases, thiA and thiB, fromClostridium acetobutylicum (Winzer et al., J. Mol. Microbiol.Biotechnol. 2:531-541 (2000)). The protein sequences for each of theseexemplary gene products can be found using the following GI numbersand/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism atoB 16130161 NP_416728.1Escherichia coli yqeF 90111494 NP_417321.2 Escherichia coli phaA113867452 YP_725941 Ralstonia eutropha bktB 3046397 AAC38322.1 Ralstoniaeutropha thiA 15896127 NP_349476.1 Clostridium acetobutylicum thiB15004782 NP_149242.1 Clostridium acetobutylicum

It is less desirable to use the thiolase-encoding genes fadA and fadB,genes in fatty acid degradation pathway in E. coli, in this exemplarypathway. These genes form a complex that encodes for multipleactivities, most of which are not desired in this pathway.

Referring to FIG. 2, step 2 involves 3-hydroxyacyl-CoA dehydrogenase.The second step in the pathway involves the reduction of 3-oxoadipyl-CoAto 3-hydroxyadipyl-CoA. The gene products encoded by phaC in Pseudomonasputida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424(1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro et al., Arch.Microbiol. 188:117-125 (2007)) catalyze the reverse reaction, that is,the oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during thecatabolism of phenylacetate or styrene. The reactions catalyzed by suchdehydrogenases are reversible and accordingly these genes representcandidates to carry out the second step of adipate synthesis as shown inFIG. 2. A similar transformation is also carried out by the gene productof hbd in Clostridium acetobutylicum (Atsumi et al., Metab. Eng. (epubSep. 14, 2007); Boynton et al., J. Bacteriol. 178:3015-3024 (1996)).This enzyme converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Lastly,given the proximity in E. coli of paaH to other genes in thephenylacetate degradation operon (Nogales et al., Microbiol. 153:357-365(2007)) and the fact that paaH mutants cannot grow on phenylacetate(Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)), it is expectedthat the E. coli paaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.The protein sequences for each of these exemplary gene products can befound using the following GI numbers and/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism paaH 16129356 NP_415913.1Escherichia coli phaC 26990000 NP_745425.1 Pseudomonas putida paaC106636095 ABF82235.1 Pseudomonas fluorescens hbd 15895965 NP_349314.1Clostridium acetobutylicum

Referring to FIG. 2, step 3 involves 3-hydroxyadipyl-CoA dehydratase.The gene product of crt from C. acetobutylicum catalyzes the dehydrationof 3-hydroxybutyryl-CoA to crotonyl-CoA (see FIG. 2) (Atsumi et al.,supra, 2007; Boynton et al., J. Bacteriol. 178:3015-3024 (1996)).Homologs of this gene are strong candidates for carrying out the thirdstep in the adipate synthesis pathway exemplified in FIG. 2. Inaddition, genes known to catalyze the hydroxylation of double bonds inenoyl-CoA compounds represent additional candidates given thereversibility of such enzymatic transformations. For example, theenoyl-CoA hydratases, phaA and phaB, of P. putida are believed to carryout the hydroxylation of double bonds during phenylacetate catabolism(Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) andthus represent additional candidates for incorporation into E. coli. Thedeletion of these genes precludes phenylacetate degradation in P.putida. The paaA and paaB from P. fluorescens catalyze analogoustransformations (Olivera et al., supra, 1998). Lastly, a number ofEscherichia coli genes have been shown to demonstrate enoyl-CoAhydratase functionality including maoC (Park and Lee, J. Bacteriol.185:5391-5397 (2003)), paaF (Ismail et al., Eur. J. Biochem.270:3047-3054 (2003); Park and Lee, Biotechnol. Bioeng. 86:681-686(2004); Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346(2004)), and paaG (Ismail et al., supra, 2003; Park and Lee, supra,2004; Park and Lee, supra, 2004). The protein sequences for each ofthese exemplary gene products can be found using the following GInumbers and/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism maoC 16129348 NP_415905.1Escherichia coli paaF 16129354 NP_415911.1 Escherichia coli paaG16129355 NP_415912.1 Escherichia coli cr 15895969 NP_349318.1Clostridium acetobutylicum paaA 26990002 NP_745427.1 Pseudomonas putidapaaB 26990001 NP_745426.1 Pseudomonas putida phaA 106636093 ABF82233.1Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonasfluorescens

Alternatively, beta-oxidation genes are candidates for the first threesteps in adipate synthesis. Candidate genes for the proposed adipatesynthesis pathway also include the native fatty acid oxidation genes ofE. coli and their homologs in other organisms. The E. coli genes fadAand fadB encode a multienzyme complex that exhibits ketoacyl-CoAthiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydrataseactivities (Yang et al., Biochem. 30:6788-6795 (1991); Yang et al., J.Biol. Chem. 265:10424-10429 (1990); Yang et al., J. Biol. Chem.266:16255 (1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937(1990)). These activities are mechanistically similar to the first threetransformations shown in FIG. 2. The fadA and fadB genes encode similarfunctions and are naturally expressed only anaerobically (Campbell etal., Mol. Microbiol. 47:793-805 (2003)). These gene products naturallyoperate to degrade short, medium, and long chain fatty-acyl-CoAcompounds to acetyl-CoA, rather than to convert succinyl-CoA andacetyl-CoA into 5-carboxy-2-pentenoyl-CoA as proposed in FIG. 2.However, it is well known that the ketoacyl-CoA thiolase,3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase enzymescatalyze reversible transformations. Furthermore, directed evolution andrelated approaches can be applied to tailor the substrate specificitiesof the native beta-oxidation machinery of E. coli. Thus these enzymes orhomologues thereof can be applied for adipate production. If the nativegenes operate to degrade adipate or its precursors in vivo, theappropriate genetic modifications are made to attenuate or eliminatethese functions. However, it may not be necessary since a method forproducing poly[(R)-3-hydroxybutyrate] in E. coli that involvesactivating fadB, by knocking out a negative regulator, fadR, andco-expressing a non-native ketothiolase, phaA from Ralstonia eutropha,has been described (Sato et al., J. Biosci. Bioeng. 103:38-44 (2007)).This work clearly demonstrated that a beta-oxidation enzyme, inparticular the gene product of fadB which encodes both 3-hydroxyacyl-CoAdehydrogenase and enoyl-CoA hydratase activities, can function as partof a pathway to produce longer chain molecules from acetyl-CoAprecursors. The protein sequences for each of these exemplary geneproducts can be found using the following GI numbers and/or GenBankidentifiers:

Gene name GI# GenBank Accession # Organism fadA 49176430 YP_026272.1Escherichia coli fadB 16131692 NP_418288.1 Escherichia coli fadI16130275 NP_416844.1 Escherichia coli fadJ 16130274 NP_416843.1Escherichia coli fadR 16129150 NP_415705.1 Escherichia coli

Referring to FIG. 2, step 4 involves 5-carboxy-2-pentenoyl-CoAreductase. Whereas the ketothiolase, dehydrogenase, and enoyl-CoAhydratase steps are generally reversible, the enoyl-CoA reductase stepis almost always oxidative and irreversible under physiologicalconditions (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).FadE catalyzes this likely irreversible transformation in E. coli(Campbell and Cronan, J. Bacteriol. 184:3759-3764 (2002)). The pathwayrequires an enzyme that can reduce a 2-enoyl-CoA intermediate, not onesuch as FadE that will only oxidize an acyl-CoA to a 2-enoyl-CoAcompound. Furthermore, although it has been suggested that E. colinaturally possesses enzymes for enoyl-CoA reduction (Mizugaki et al., J.Biochem. 92:1649-1654 (1982); Nishimaki et al., J. Biochem. 95:1315-1321(1984)), no E. coli gene possessing this function has been biochemicallycharacterized.

One candidate gene for the enoyl-CoA reductase step is the gene productof bcd from C. acetobutylicum (Atsumi et al., supra, 2007; Boynton etal., J. Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes thereduction of crotonyl-CoA to butyryl-CoA, a reaction similar inmechanism to the desired reduction of 5-carboxy-2-pentenoyl-CoA toadipyl-CoA in the adipate synthesis pathway. Activity of this enzyme canbe enhanced by expressing bcd in conjunction with expression of the C.acetobutylicum etfAB genes, which encode an electron transferflavoprotein. An additional candidate for the enoyl-CoA reductase stepis the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeisteret al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived fromthis sequence following the removal of its mitochondrial targetingleader sequence was cloned in E. coli, resulting in an active enzyme(Hoffmeister et al., supra, 2005). This approach is well known to thoseskilled in the art of expressing eukarytotic genes, particularly thosewith leader sequences that may target the gene product to a specificintracellular compartment, in prokaryotic organisms. A close homolog ofthis gene, TDE0597, from the prokaryote Treponema denticola represents athird enoyl-CoA reductase which has been cloned and expressed in E. coli(Tucci and Martin, FEBS Lett. 581:1561-1566 (2007)). The proteinsequences for each of these exemplary gene products can be found usingthe following GI numbers and/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism bcd 15895968 NP_349317.1Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridiumacetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum TER62287512 Q5EU90.1 Euglena gracilis TDE0597 42526113 NP_971211.1Treponema denticola

Referring to FIG. 2, step 5 involves adipyl-CoA synthetase (alsoreferred to as adipate-CoA ligase), phosphotransadipylase/adipatekinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. Froman energetic standpoint, it is desirable for the final step in theadipate synthesis pathway to be catalyzed by an enzyme or enzyme pairthat can conserve the ATP equivalent stored in the thioester bond ofadipyl-CoA. The product of the sucC and sucD genes of E. coli, orhomologs thereof, can potentially catalyze the final transformationshown in FIG. 2 should they exhibit activity on adipyl-CoA. The sucCDgenes naturally form a succinyl-CoA synthetase complex that catalyzesthe formation of succinyl-CoA from succinate with the concaminantconsumption of one ATP, a reaction which is reversible in vivo (Buck etal., Biochem. 24:6245-6252 (1985)). Given the structural similaritybetween succinate and adipate, that is, both are straight chaindicarboxylic acids, it is reasonable to expect some activity of thesucCD enzyme on adipyl-CoA. An enzyme exhibiting adipyl-CoA ligaseactivity can equivalently carry out the ATP-generating production ofadipate from adipyl-CoA, here using AMP and PPi as cofactors, whenoperating in the opposite physiological direction as depicted in FIG. 1.Exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for whichthe sequence is yet uncharacterized (Vamecq et al., Biochem. J.230:683-693 (1985)), either of the two characterized phenylacetate-CoAligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395,147-155 (2005); Wang et al., Biochem. Biophy. Res. Commun. 360:453-458(2007)), the phenylacetate-CoA ligase from Pseudomonas putida(Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al., J.Bacteriol. 178:4122-4130 (1996)). The protein sequences for each ofthese exemplary gene products can be found using the following GInumbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism sucC 16128703 NP_415256.1Escherichia coli sucD 1786949 AAC73823.1 Escherichia coli

Another option, using phosphotransadipylase/adipate kinase, is catalyzedby the gene products of buk1, buk2, and ptb from C. acetobutylicum(Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol.Microbiol. Biotechnol. 2:33-38 (2000)), or homologs thereof. The ptbgene encodes an enzyme that can convert butyryl-CoA intobutyryl-phosphate, which is then converted to butyrate via either of thebuk gene products with the concomitant generation of ATP. The analogousset of transformations, that is, conversion of adipyl-CoA toadipyl-phosphate followed by conversion of adipyl-phosphate to adipate,can be carried out by the buk1, buk2, and ptb gene products. The proteinsequences for each of these exemplary gene products can be found usingthe following GI numbers and/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism ptb 15896327 NP_349676Clostridium acetobutylicum buk1 15896326 NP_349675 Clostridiumacetobutylicum buk2 20137415 Q97II1 Clostridium acetobutylicum

Alternatively, an acetyltransferase capable of transferring the CoAgroup from adipyl-CoA to acetate can be applied. Similar transformationsare catalyzed by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity,respectively (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). Theprotein sequences for each of these exemplary gene products can be foundusing the following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3146349050 EDK35586.1 Clostridium kluyveri

Finally, though not as desirable from an energetic standpoint, theconversion of adipyl-CoA to adipate can also be carried out by anacyl-CoA hydrolase or equivalently a thioesterase. The top E. coli genecandidate is tesB (Naggert et al., J. Biol. Chem. 266:11044-11050(1991)), which shows high similarity to the human acot8, which is adicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westinet al., J. Biol. Chem. 280:38125-38132 (2005)). This activity has alsobeen characterized in the rat liver (Deana, Biochem. Int. 26:767-773(1992)). The protein sequences for each of these exemplary gene productscan be found using the following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism tesB 16128437 NP_414986Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669NP_570112 Rattus norvegicus

Other native candidate genes include tesA (Bonner and Bloch, J. Biol.Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol.Rev. 29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)),paaI (Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB(Leduc et al., J. Bacteriol. 189:7112-7126 (2007)). The proteinsequences for each of these exemplary gene products can be found usingthe following GI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism tesA 16128478 NP_415027Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paaI 16129357NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli

The above description provides an exemplary adipate synthesis pathway byway of a reverse adipate degradation pathway.

Example II Preparation of an Adipate Producing Microbial Organism Havinga Reverse Degradation Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using the reverse degradation pathway.

Escherichia coli is used as a target organism to engineer a reverseadipate degradation pathway as shown in FIG. 2. E. coli provides a goodhost for generating a non-naturally occurring microorganism capable ofproducing adipate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleicacids encoding the enzymes utilized in the reverse degradation pathwayare expressed in E. coli using well known molecular biology techniques(see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). Inparticular, the paaI (NP 415915.1), paaH (NP 415913.1), and maoC (NP415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydrataseactivities, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd(NP_349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 andAAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase andadipyl-CoA synthetase activities, respectively, are cloned into thepZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The two sets of plasmids are transformed into E. coli strainMG1655 to express the proteins and enzymes required for adipatesynthesis via the reverse degradation pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression ofreverse degradation pathway genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produceadipate is confirmed using HPLC, gas chromatography-mass spectrometry(GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of adipate. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of adipate.Adaptive evolution also can be used to generate better producers of, forexample, the acetyl-CoA and succinyl-CoA intermediates or the adipateproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the adipate producer to further increaseproduction.

For large-scale production of adipate, the above reverse degradationpathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing the culture vessel, forexample, flasks can be sealed with a septum and crimp-cap. Microaerobicconditions also can be utilized by providing a small hole in the septumfor limited aeration. The pH of the medium is maintained at a pH ofaround 7 by addition of an acid, such as H₂SO₄. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm) and the glucose uptake rate by monitoring carbon source depletionover time. Byproducts such as undesirable alcohols, organic acids, andresidual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), forexample, using an Aminex® series of HPLC columns (for example, HPX-87series) (BioRad, Hercules Calif.), using a refractive index detector forglucose and alcohols, and a UV detector for organic acids (Lin et al.,Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of an adipate producing microbialorganism using a reverse degradation pathway.

Example III Adipate Synthesis Through 3-Oxoadipate

This example describes an exemplary adipate synthesis pathway through3-oxoadipate.

An additional pathway from that described in Examples I and II that usesacetyl-CoA and succinyl-CoA as precursors for adipate formation andpasses through the metabolic intermediate, 3-oxoadipate, is shown inFIG. 3. The initial two transformations in this pathway are the twoterminal steps of the degradation pathway for aromatic andcholoroaromatic compounds operating in the reverse direction (Kaschabeket al., J. Bacteriol. 184:207-215 (2002); Nogales et al., Microbiol.153:357-365 (2007); Ismail et al., Eur. J. Biochem. 270:3047-3054(2003)). Specifically, the first step forms 3-oxoadipyl CoA by thecondensation of succinyl- and acetyl-CoA. The second step forms3-oxoadipate and is reported to be reversible in Pseudomonas sp. StrainB13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)).

The subsequent steps involve reduction of 3-oxoadipate to3-hydroxyadipate (conversion of a keto group to hydroxyl group),dehydration of 3-hydroxyadipate to yield hexa-2-enedioate, and reductionof hexa-2-enedioate to form adipate. These steps of the pathway areanalogous to the conversion of oxaloacetate into succinate via thereductive TCA cycle (see FIG. 4). This supports the steps in the pathwaybeing thermodynamically favorable subject to the presence of appropriatemetabolite concentrations. The final reduction step can be carried outeither biochemically or by employing a chemical catalyst to converthexa-2-enedioate into adipate. Chemical hydrogenation can be performedusing Pt catalyst on activated carbon as has been described in (Niu etal., Biotechnol. Prog. 18:201-211 (2002)).

The maximum theoretical yield of adipate using this pathway is 0.92 moleper mole glucose consumed, and oxygen is not required for attainingthese yields (see Table 2). The associated energetics are identical tothose of the reverse adipate pathway. Theoretically, ATP formation of upto 1.55 moles is observed per mole of glucose utilized through thispathway. The ATP yield improves to approximately 2.47 moles ifphosphoenolpyruvate kinase (PPCK) is assumed to operate in the directionof ATP generation. Interestingly, the product yield can be increasedfurther to 1 mole adipate per mole of glucose consumed if chemicalhydrogenation is used for the last step and a 100% efficiency ofcatalysis is assumed. In this scenario, up to 1.95 moles of ATP areformed theoretically without assuming the reverse functionality of PPCK.

TABLE 2 The maximum theoretical yields of adipate and the associated ATPyields per mole of glucose using the 3-oxoadipate pathway. Final stepFinal step chemical enzymatic hydrogenation Aerobic Anaerobic AerobicAnaerobic Adipate Yield 0.92 0.92 1.00 1.00 Max ATP yield @ max 1.551.55 1.95 1.95 adipate yield

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of adipate, one or moreexogenous DNA sequence(s) can be expressed in a host microorganism. Inaddition, the host microorganism can have endogenous gene(s)functionally deleted. These modifications allow the production ofadipate using renewable feedstock.

Described below are a number of biochemically characterized candidategenes capable of encoding enzymes that catalyze each step of the3-oxoadipate pathway for adipate synthesis. Although this method isdescribed for E. coli, one skilled in the art can apply these teachingsto any other suitable host organism. Specifically, listed below aregenes that are native to E. coli as well as genes in other organismsthat can be applied to catalyze the appropriate transformations whenproperly cloned and expressed.

Referring to FIG. 3, step 1 involves succinyl CoA:acetyl CoA acyltransferase (β-ketothiolase). Gene candidates for this enzyme are listedabove (FIG. 2, step 1).

Referring to FIG. 3, step 2 involves 3-oxoadipyl-CoA transferase. Inthis step, 3-oxoadipate is formed by the transfer of the CoA group from3-oxoadipyl-CoA to succinate. This activity is reported in a two-unitenzyme encoded by pcaI and pcaJ in Pseudomonas (Kaschabek et al., J.Bacteriol. 184:207-215 (2002)). This enzyme catalyzes a reversibletransformation. The protein sequences of exemplary gene products forsubunit A of this complex can be found using the following GI numbersand/or GenBank identifiers:

GenBank Gene name GI# Accession # Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaI 50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaI21224997 NP_630776.1 Streptomyces coelicolor

The protein sequences of exemplary gene products for subunit B of thiscomplex can be found using the following GI numbers and/or GenBankidentifiers:

GenBank Gene name GI# Accession # Organism pcaJ 26990657 NP_746082.1Pseudomonas putida pcaJ 21224996 NP_630775.1 Streptomyces coelicolorpcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1

Referring to FIG. 3, step 3 involves 3-oxoadipate reductase. E. coli hasseveral candidate alcohol dehydrogenases; two that have analogousfunctions are malate dehydrogenase (mdh) and lactate dehydrogenase(ldhA). While it has not been shown that these two enzymes have broadsubstrate specificities in E. coli, lactate dehydrogenase from Ralstoniaeutropha has been shown to demonstrate high activities on substrates ofvarious chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoateand 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J. Biochem.130:329-334 (1983)). An additional non-native enzyme candidate for thisstep is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from thehuman heart which has been cloned and characterized (Marks et al., J.Biol. Chem. 267:15459-15463 (1992)). This enzyme is particularlyinteresting in that it is a dehydrogenase that operates on a3-hydroxyacid. Given that dehydrogenases are typically reversible, it isexpected that this gene product, or a homlog thereof, will be capable ofreducing a 3-oxoacid, for example, 3-oxoadipate, to the corresponding3-hydroxyacid, for example, 3-hydroxyadipate. The protein sequences foreach of these exemplary gene products can be found using the followingGI numbers and/or GenBank identifiers:

Gene name GI# GenBank Accession # Organism mdh 1789632 AAC76268.1Escherichia coli ldhA 16129341 NP_415898.1 Escherichia coli ldh113866693 YP_725182.1 Ralstonia eutropha bdh 177198 AAA58352.1 Homosapiens

Referring to FIG. 3, step 4 involves 3-hydroxyadipate dehydratase. Inthis reaction, 3-hydroxyadipate is dehydrated to hexa-2-enedioate.Although no direct evidence for this enzymatic transformation has beenidentified, most dehydratases catalyze the α, β-elimination of water.This involves activation of the α-hydrogen by an electron-withdrawingcarbonyl, carboxylate, or CoA-thiol ester group and removal of thehydroxyl group from the β-position (Martins et al., Proc. Natl. Acad.Sci. USA 101:15645-15649 (2004); Buckel and Golding, FEMS Microbiol.Rev. 22:523-541 (1998)). The protein sequences for exemplary geneproducts can be found using the following GI numbers and/or GenBankidentifiers:

Gene name GI# GenBank Accession # Organism acnA 166215073 P25516.3Escherichia coli fumB 33112655 P14407.2 Escherichia coli ilvD 146451AAA24013.1 Escherichia coli

Other good candidates for carrying out this function are the serinedehydratases. These enzymes catalyze a very similar transformation inthe removal of ammonia from serine as required in this dehydration step.The protein sequence for exemplary gene product can be found using thefollowing GI number and/or GenBank identifier:

Gene name GI# GenBank Accession # Organism dsdA 2507445 P00926Escherichia coli

Non-native gene candidates for this transformation have been identifiedas well. For example, the multi-subunit L-serine dehydratase fromPeptostreptococcus asaccharolyticus was shown to complement an E. colistrain deficient in L-serine dehydratase activity (Hofmeister et al., J.Bacteriol. 179:4937-4941 (1997)). Further, a putative2-(hydroxymethyl)glutarate dehydratase, encoded by the gene hmd inEubacterium barkeri shows similarity to both α- and β-subunits of[4Fe-4S]-containing bacterial serine dehydratases (Alhapel et al., Proc.Natl. Acad. Sci. USA 103:12341-12346 (2006)). The protein sequence forexemplary gene product can be found using the following GI number and/orGenBank identifier:

Gene name GI# GenBank Accession # Organism Hmd 86278275 ABC88407.1Eubacterium barkeri

Referring to FIG. 3, step 5 involves 2-enoate reductase. The final stepin the 3-oxoadipate pathway is reduction of the double bond inhexa-3-enedioate to form adipate. Biochemically, this transformation canbe catalyzed by 2-enoate reductase (EC 1.3.1.31) known to catalyze theNADH-dependent reduction of a wide variety of α, β-unsaturatedcarboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem.276:5779-5787 (2001)). This enzyme is encoded by enr in several speciesof Clostridia (Giesel and Simon, Arch. Microbiol. 135:51-57 (1983))including C. tyrobutyricum and C. thermoaceticum (now called Moorellathermoaceticum) (Rohdich, et al., J. Biol. Chem. 276:5779-5787 (2001)).In the recently published genome sequence of C. kluyveri, 9 codingsequences for enoate reductases have been reported, out of which one hasbeen characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and C.thermoaceticum have been cloned and sequenced and show 59% identity toeach other. The former gene is also found to have approximately 75%similarity to the characterized gene in C. kluyveri (Giesel and Simon,Arch. Microbiol. 135:51-57 (1983)). It has been reported based on thesesequence results that enr is very similar to the dienoyl CoA reductasein E. coli (fadH) (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)).Several gene candidates thus exist for catalyzing this last step in the3-oxoadipate pathway and have been listed below. The C. thermoaceticumenr gene has also been expressed in an enzymatically active form in E.coli (Rohdich et al., supra, 2001). The protein sequences for exemplarygene products can be found using the following GI numbers and/or GenBankidentifiers:

Gene GenBank name GI# Accession # Organism fadH 16130976 NP_417552.1Escherichia coli enr 169405742 ACA54153.1 Clostridium botulinum A3 strenr 2765041 CAA71086.1 Clostridium tyrobutyricum enr 3402834 CAA76083.1Clostridium kluyveri

The above description provides an exemplary adipate synthesis pathway byway of an 3-oxoadipate pathway.

Example IV Preparation of an Adipate Producing Microbial Organism Havinga 3-Oxoadipate Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using the 3-oxoadipate pathway.

Escherichia coli is used as a target organism to engineer the3-oxoadipate pathway as shown in FIG. 3. E. coli provides a good hostfor generating a non-naturally occurring microorganism capable ofproducing adipate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleicacids encoding the enzymes utilized in the 3-oxoadipate pathway areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular,the paaJ (NP_415915.1), pcaIJ (AAN69545.1 and NP_746082.1), and bdh(AAA58352.1) genes encoding the succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, and 3-oxoadipate reductaseactivities, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the acnA(P25516.3) and enr (ACA54153.1) genes encoding 3-hydroxyadipatedehydratase and 2-enoate reductase activities, respectively, are clonedinto the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The two sets of plasmids are transformed into E. coli strainMG1655 to express the proteins and enzymes required for adipatesynthesis via the 3-oxoadipate pathway.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the3-oxoadipate pathway genes for adipate synthesis is corroborated usingmethods well known in the art for determining polypeptide expression orenzymatic activity, including for example, Northern blots, PCRamplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce adipate is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of adipate. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of adipate.Adaptive evolution also can be used to generate better producers of, forexample, the acetyl-CoA and succinyl-CoA intermediates or the adipateproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the adipate producer to further increaseproduction.

For large-scale production of adipate, the 3-oxoadipatepathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing the culture vessel, forexample, flasks can be sealed with a septum and crimp-cap. Microaerobicconditions also can be utilized by providing a small hole in the septumfor limited aeration. The pH of the medium is maintained at around a pHof 7 by addition of an acid, such as H₂SO₄. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm) and the glucose uptake rate by monitoring carbon source depletionover time. Byproducts such as undesirable alcohols, organic acids, andresidual glucose can be quantified by HPLC (Shimadzu), for example,using an Aminex® series of HPLC columns (for example, HPX-87 series)(BioRad), using a refractive index detector for glucose and alcohols,and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng.775-779 (2005)).

This example describes the preparation of an adipate-producing microbialorganism containing a 3-oxidoadipate pathway.

Example V Adipate Synthesis Via Cis,Cis-Muconic Acid

This example describes an adipate synthesis pathway previously described(see Niu et al., Biotechnol. Prog. 18(2): p. 201-11. 2002; Frost et al.,U.S. Pat. No. 5,487,987, issued Jan. 30, 1996).

Adipate synthesis via a combined biological and chemical conversionprocess has been previously described. (Niu et al., Biotechnol. Prog.18:201-211 (2002)) and is shown in FIG. 5. This method is furtherdescribed in U.S. Pat. No. 5,487,987. Adipate synthesis through thisroute entails introduction of three heterologous genes into E. coli thatcan convert dehydroshikimate into cis,cis-muconic acid (Niu et al.,supra, 2002). A final chemical hydrogenation step leads to the formationof adipic acid. In this step, the pretreated fermentation broth thatcontained 150 mM cis,cis-muconate was mixed with 10% platinum (Pt) onactivated carbon. The hydrogenation reaction was carried out at 3400 KPaof hydrogen pressure for two and a half hour at 250° C. with stirring.The calculated adipate yields are shown in Table 3 assuming either anenzymatic or chemical catalysis step is utilized to convertcis,cis-muconate into adipate. Under aerobic conditions, an 85% molaryield of adipate can be obtained if a chemical reaction is employed forhydrogenation and a 75% molar yield is obtained if an NADH-basedhydrogenase is used.

TABLE 3 The maximum theoretical yields of adipate per mole of glucoseusing the using the cis,cis-muconic acid pathway. Final step chemicalFinal step enzymatic hydrogenation Aerobic Anaerobic Aerobic AnaerobicAdipate Yield 0.75 0.00 0.85 0.00

Although this is an exemplary method, there are disadvantages of thismethod compared to others, such as those described in Examples I-IV. Forexample, the first limitation of this method is the lower theoreticalyields compared to the reverse adipate degradation and 3-oxoadipatepathways. The second limitation is that the ATP yields of this pathwayare negligible. A third limitation of this pathway is that it involves adioxygenase, necessitating a supply of oxygen to the bioreactor andprecluding the option of anaerobic fermentation.

The above description provides an exemplary adipate synthesis pathway byway of a cis,cis-muconic acid pathway

Example VI Adipate Synthesis Via Alpha-Ketoadipate

This example describes an exemplary adipate synthesis pathway via analpha-ketoadipate pathway.

Alpha-keto adipate is a known intermediate in lysine biosynthesis in S.cerevisiae, and this information was used to identify an additionalpathway for adipic acid biosynthesis (see FIG. 6). Conversion ofalpha-ketoglutarate to alpha-ketoadipate is catalyzed by homocitratesynthase, homoaconitase, and homoisocitrate dehydrogenase as indicatedby dashed arrows in FIG. 6. Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.,Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys. Res. Commun. 77:586-591 (1977). Subsequent steps involve adehydratase for the conversion of alpha-hydroxyadipate intohexa-2-enedioate followed by its reduction to adipic acid. This laststep can be catalyzed either by an enzyme or can take place through achemical reaction as described in Example II. Genes encoding the enzymesfor the alpha-ketoadipate pathway are identified as described inExamples I-IV.

The adipate yields associated with this pathway are shown in Table 4.Because of the loss of two CO₂ molecules during the conversion ofacetyl-CoA to adipate, only 67% of the glucose can be converted intoadipate. This is reflected in the molar yields for this pathway underaerobic conditions. The yields are further reduced in the absence ofoxygen uptake. Also since the maximum ATP yields under anaerobicconditions are negligible, the engineered organism will have to utilizeadditional substrate to form energy for cell growth and maintenanceunder such conditions.

TABLE 4 The maximum theoretical yields of adipate and the associated ATPyields per mole of glucose using the using the alpha-ketoadipatepathway. Final step Final step chemical enzymatic hydrogenation AerobicAnaerobic Aerobic Anaerobic Adipate Yield 0.67 0.45 0.67 0.40 Max ATPyield @ max 6.17 0.00 7.50 0.00 adipate yield

The above description provides an exemplary adipate synthesis pathway byway of an alpha-ketoadipate pathway.

Example VII Adipate Synthesis Via Lysine Degradation

This example describes an exemplary adipate synthesis pathway via alysine degradation pathway.

Two additional pathways for adipate synthesis rely on lysine degradationto form adipate. One pathway starts from alpha-ketoglutarate to formlysine (pathway non-native to E. coli and found in S. cerevisiae), andthe other uses aspartate as a starting point for lysine biosynthesis(pathway native to E. coli). FIG. 7 shows adipate formation from lysine.The maximum theoretical yields for adipate, both in the presence andabsence of oxygen, using the E. coli stoichiometric model are shown inTables 5 and 6, with alpha-ketoglutarate and aspartate as the respectivestarting points for lysine. The maximum ATP yields accompanying thesetheoretical yields were also calculated and are shown in the sametables. These yields are lower in comparison to the other pathwaysdescribed in Examples I-IV. Genes encoding the enzymes for thealpha-ketoadipate pathway are identified as described in Examples I-IV.

TABLE 5 The maximum theoretical yield of adipate and the accompanyingATP yield per mole of glucose assuming the lysine biosynthesis pathwaywith alpha-ketoglutarate as a starting point. Aerobic Anaerobic AdipateYield 0.40 0.20 Max ATP yield @ max adipate yield 5.60 0.00

TABLE 6 The maximum theoretical yield of adipate and the accompanyingATP yield per mole of glucose assuming the lysine biosynthesis pathwaywith aspartate as a starting point. Aerobic Anaerobic Adipate Yield 0.500.34 Max ATP yield @ max adipate yield 0.50 0.04

The above description provides an exemplary adipate synthesis pathway byway of a lysine degradation pathway.

Example VIII Production of Caprolactam and 6-Aminocaproic Acid ViaAdipyl-CoA

This example describes an exemplary caprolactam and/or 6-aminocaproicacid synthesis pathway via an adipyl-CoA pathway.

An exemplary pathway for forming caprolactam and/or 6-aminocaproic acidusing adipyl-CoA as the precursor is shown in FIG. 8. The pathwayinvolves a CoA-dependant aldehyde dehydrogenase that can reduceadipyl-CoA to adipate semialdehyde and a transaminase or 6-aminocaproatedehydrogenase that can transform this molecule into 6-aminocaproic acid.The terminal step that converts 6-aminocaproate into caprolactam can beaccomplished either via an amidohydrolase or via chemical conversion(Guit and Buijs, U.S. Pat. No. 6,353,100, issued Mar. 7, 2002; Wolterset al., U.S. Pat. No. 5,700,934, issued Dec. 23, 1997; Agterberg et al.,U.S. Pat. No. 6,660,857, issued Dec. 9, 2003). The maximum theoreticalyield of caprolactam was calculated to be 0.8 mole per mole glucoseconsumed (see Table 7) assuming that the reverse adipate degradationpathway was complemented with the reaction scheme shown in FIG. 8. Thepathway is favorable energetically as up to 0.78 moles of ATP are formedper mole of glucose consumed at the maximum theoretical yield ofcaprolactam. The ATP yield can be further improved to 1.63 moles of ATPproduced per mole of glucose if phosphoenolpyruvate carboxykinase (PPCK)is assumed to function in the ATP-generating direction towardsoxaloacetate formation.

The final amidohydrolase step is energetically and redox neutral, andthus the product and ATP molar yields associated with 6-aminocaproicacid production are equivalent to those associated with caprolactamproduction. Thus one can alternatively envision a microorganism andassociated fermentation process that forms 6-aminocaproic acid insteadof caprolactam followed by an additional unit operation todehydrate/cyclize 6-aminocaproic acid to caprolactam.

TABLE 7 The maximum theoretical yield of caprolactam and theaccompanying ATP yield per mole of glucose assuming that the reversefatty acid degradation pathway is complemented with the reaction schemefrom FIG. 8. Aerobic Anaerobic Caprolactam Yield 0.80 0.80 Max ATP yield@ max Caprolactam yield 0.78 0.78 Max ATP yield @ max Caprolactam yield1.63 1.63 PPCK assumed

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of 6-aminocaproic acidor caprolactam, one or more exogenous DNA sequence(s) can be expressedin a host microorganism. In addition, the microorganism can haveendogenous gene(s) functionally deleted. These modifications will allowthe production of 6-aminocaproate or caprolactam using renewablefeedstock.

Below is described a number of biochemically characterized candidategenes capable of encoding enzymes that catalyze each step of thecaprolactam formation pathway described in FIG. 8. Although describedfor E. coli, one skilled in the art can apply these teachings to anyother suitable host organism. Specifically, the genes listed are nativeto E. coli or are genes in other organisms that can be applied tocatalyze the appropriate transformations when properly cloned andexpressed.

Referring to FIG. 8, step 1 involves CoA-dependant aldehydedehydrogenase. Exemplary genes that encode enzymes for catalyzing thereduction of an acyl-coA to its corresponding aldehyde include theAcinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase(Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997)), theAcinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)) and the sucD gene fromClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)), which can convert succinyl-CoA to succinate semialdehyde.

Gene GenBank name GI# Accession # Organism acr1 50086359 YP_047869.1Acinetobacter calcoaceticus 18857901 BAB85476.1 Acinetobacter sp. StrainM-1 sucD 172046062 P38947.1 Clostridium kluyveri

Referring to FIG. 8, step 2 involves transaminase. The second step inthe pathway is conversion of the 6-aldehyde to an amine. Thistransformation can likely be accomplished by gamma-aminobutyratetransaminase (GABA transaminase), a native enzyme encoded by gabT thattransfers an amino group from glutamate to the terminal aldehyde ofsuccinyl semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042(1990)). The gene product of puuE catalyzes another 4-aminobutyratetransaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608(2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens,and Sus scrofa have been shown to react with 6-aminocaproic acid(Cooper, Methods Enzymol. 113:80-82 (1985); Scott and Jakoby, J. Biol.Chem. 234:932-936 (1959)). The protein sequences for exemplary geneproducts can be found using the following GI numbers and/or GenBankidentifiers:

GenBank Gene name GI# Accession # Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Referring to FIG. 8, step 2 can alternatively involve 6-aminocaproatedehydrogenase which comprises the reductive amination of adipatesemialdehyde to form 6-aminocaproate. This transformation can beaccomplished by lysine-6-dehydrogenase, which naturally convertsL-lysine to 2-aminoadipate-6-semialdehyde. Exemplary enzymes can befound in Geobacillus stearothermophilus (Heydari et al., Appl. Environ.Microbiol. 70(2):937-942 (2004)), Agrobacterium tumefaciens (Hashimotoet al., J. Biochem. (Tokyo), 106(1):76-80 (1989); Misono et al., J.Biochem. (Tokyo), 105(6):1002-1008 (1989)), and Achromobacterdenitrificans (Ruldeekulthamrong et al., BMB Reports 790-795 (2008)).

GenBank Gene name GI# Accession # Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

Referring to FIG. 8, step 3 involves amidohydrolase. The final step ofcaprolactam synthesis is cyclization of 6-aminocaproic acid. Thistransformation has not been characterized enzymatically but it is verysimilar to the cyclization of lysine by D-lysine lactamase (EC 3.5.2.11)from Cryptococcus laurentii (Fukumura et al., FEBS Lett. 89:298-300(1978)). However, the protein and nucleotide sequences of this enzymeare not currently known and, so far, lysine lactamase activity has notbeen demonstrated in other organisms.

Plasmids contained in several strains of Pseudomonas sp. isolated fromsoil have been shown to confer ability to grow on caprolactam as a solecarbon source (Boronin et al., FEMS Microbiol. Lett. 22:167-170 (1984));however, associated gene or protein sequences have not been associatedwith this function to date.

The most closely related candidate enzyme with available sequenceinformation is 6-aminohexanoate-cyclic dimer hydrolase, which has beencharacterized in Pseudomonas sp. and Flavobacterium sp. The nylB geneproduct from Pseudomonas sp NK87 was cloned and expressed in E. coli(Kanagawa et al., J. Gen. Microbiol. 139:787-795 (1993)). The substratespecificity of the enzyme was tested in Flavobacterium sp K172 and wasshown to react with higher-order oligomers of 6-aminohexanoate but notcaprolactam (Kinoshita et al., Eur. J. Biochem. 116:547-551 (1981)). Thereversibility and ability of 6-aminohexanoate dimer hydrolases in otherorganisms to react with the desired substrate in the direction ofinterest can be further tested. The protein sequences for exemplary geneproducts can be found using the following GI numbers and/or GenBankidentifiers:

Gene GenBank name GI# Accession # Organism nylB 148711 AAA24929.1Pseudomonas sp NK87 nylB 129000 P13397 Flavobacterium sp K172 nylB119961013 YP_949627.1 Arthrobacter aurescens TC1

The above description provides an exemplary pathway to producecaprolactam and/or 6-aminocaproic acid by way of an adipyl-CoA pathway.

Example IX Preparation of a 6-Aminocaproate or Caprolactam ProducingMicrobial Organism Having a 3-Oxoadipate Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using the reverse degradation pathway and convertingthe intracellular adipate to 6-aminocaproate and/or caprolactam.

Escherichia coli is used as a target organism to engineer the necessarygenes for adipate, 6-aminocaproate, and/or caprolactam synthesis (seeFIG. 2 and FIG. 8). E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing adipate,6-aminocaproate, and/or caprolactam. E. coli is amenable to geneticmanipulation and is known to be capable of producing various products,like ethanol, acetic acid, formic acid, lactic acid, and succinic acid,effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproateand/or caprolactam, nucleic acids encoding the enzymes utilized in thereverse adipate degradation pathway and 6-aminocaproate or caprolactamsynthesis pathways are expressed in E. coli using well known molecularbiology techniques (see, for example, Sambrook, supra, 2001; Ausubel,supra, 1999). In particular, the paaJ (NP_415915.1), paaH (NP_415913.1),and maoC (NP_415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoAdehydratase activities, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Inaddition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1), andsucCD (NP_415256.1 and AAC73823.1) genes encoding5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetaseactivities, respectively, are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the acr1(YP_047869.1), gabT (NP_417148.1), and nylB (AAA24929.1) genes encodingCoA-dependent aldehyde dehydrogenase, transaminase, and amidohydrolaseactivities are cloned into a third compatible plasmid, pZS23, under thePA1/lacO promoter. pZS23 is obtained by replacing the ampicillinresistance module of the pZS13 vector (Expressys, Ruelzheim, Germany)with a kanamycin resistance module by well-known molecular biologytechniques. The three sets of plasmids are transformed into E. colistrain MG1655 to express the proteins and enzymes required for6-aminocaproate and/or caprolactam synthesis.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the6-aminocaproate and caprolactam synthesis genes is corroborated usingmethods well known in the art for determining polypeptide expression orenzymatic activity, including for example, Northern blots, PCRamplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce 6-aminocaproate and/or caprolactam is confirmed usingHPLC, gas chromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional pathway for thesynthesis of 6-aminocaproate and/or caprolactam are further augmented byoptimization for efficient utilization of the pathway. Briefly, theengineered strain is assessed to determine whether any of the exogenousgenes are expressed at a rate limiting level. Expression is increasedfor any enzymes expressed at low levels that can limit the flux throughthe pathway by, for example, introduction of additional gene copynumbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 6-aminocaproate and/or caprolactam.One modeling method is the bilevel optimization approach, OptKnock(Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which isapplied to select gene knockouts that collectively result in betterproduction of 6-aminocaproate and/or caprolactam. Adaptive evolutionalso can be used to generate better producers of, for example, theacetyl-CoA and succinyl-CoA intermediates of the products. Adaptiveevolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the 6-aminocaproate and/or caprolactamproducer to further increase production.

For large-scale production of 6-aminocaproate and/or caprolactam, theabove organism is cultured in a fermenter using a medium known in theart to support growth of the organism under anaerobic conditions.Fermentations are performed in either a batch, fed-batch or continuousmanner. Anaerobic conditions are maintained by first sparging the mediumwith nitrogen and then sealing the culture vessel, for example, flaskscan be sealed with a septum and crimp-cap. Microaerobic conditions alsocan be utilized by providing a small hole in the septum for limitedaeration. The pH of the medium is maintained at around a pH of 7 byaddition of an acid, such as H₂SO₄. The growth rate is determined bymeasuring optical density using a spectrophotometer (600 nm) and theglucose uptake rate by monitoring carbon source depletion over time.Byproducts such as undesirable alcohols, organic acids, and residualglucose can be quantified by HPLC (Shimadzu), for example, using anAminex® series of HPLC columns (for example, HPX-87 series) (BioRad),using a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779(2005)).

Example X Adipate Synthesis Via 2-Hydroxyadipyl-CoA

This example describes two exemplary adipate synthesis pathwaysproceeding from alpha-ketoadipate and passing through a2-hydroxyadipyl-CoA intermediate.

As described in example VI, alpha-ketoadipate is a known intermediate inlysine biosynthesis that can be formed from alpha-ketoglutarate viahomocitrate synthase, homoaconitase, and homoisocitrate dehydrogenase.Alpha-ketoadipate can be converted to 2-hydroxyadipyl-CoA by the tworoutes depicted in FIG. 9. 2-hydroxyadipyl-CoA can be subsequentlydehydrated and reduced to adipyl-CoA which can then be converted toadipate as shown in FIG. 9. The maximum yield of adipate from glucosevia these pathways is 0.67 mol/mol.

Conversion of alpha-ketoadipate into 2-hydroxyadipate can be catalyzedby 2-ketoadipate reductase, an enzyme reported to be found in rat and inhuman placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976);Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977).Alternatively, enzymes capable of reducing alpha-ketoglutarate to2-hydroxyglutarate may also show activity on alpha-ketoadipate, which isonly one carbon atom longer. One such enzyme possessingalpha-ketoglutarate reductase activity is serA of Escherichia coli (Zhaoand Winkler, J. Bacteriol. 178(1):232-9 (1996)). Additional exemplaryenzymes can be found in Arabidopsis thaliana (Ho, et al., J. Biol. Chem.274(1):397-402 (1999)) and Haemophilus influenzae.

Gene name GI# GenBank Accession # Organism serA 16130814 NP_417388.1Escherichia coli PGDH 18394525 NP_564034 Arabidopsis thaliana serA1173427 P43885 Haemophilus influenzae

Referring to FIG. 9, 2-hydroxyadipate can likely be converted to2-hydroxyadipyl-CoA by the synthetases, transferases,phosphotransadipylases and kinases described in example I.Alternatively, enzymes with 2-hydroxyglutarate CoA-transferase orglutaconate CoA-transferase activity are likely suitable to transfer aCoA moiety to 2-hydroxyadipate. One example of such an enzyme is encodedby the gctA and gctB genes of Acidaminococcus fermentans (Buckel, etal., Eur. J. Biochem. 118(2):315-321 (1981); Mack, et al., Eur. J.Biochem. 226(1):41-51 (1994)). Similarly, synthetase, transferase, orphosphotransadipylase and kinase activities would be required to convertalpha-ketoadipate into alpha-ketoadipyl-CoA, as depicted in FIG. 9.Conversion of alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA can be carriedout by an alpha-hydroxyacyl-CoA dehydrogenase enzyme. A similar activitywas reported in propionate-adapted E. coli cells whose extractscatalyzed the oxidation of lactyl-CoA to form pyruvyl-CoA (Megraw etal., J. Bacteriol. 90(4): 984-988 (1965)). Additional hydroxyacyl-CoAdehydrogenases were described in example I.

Gene name GI# GenBank Accession # Organism gctA 3122155 Q59111Acidaminococcus fermentans gctB 3122156 Q59112 Acidaminococcusfermentans

The dehydration of 2-hydroxyadipyl-CoA to form 5-carboxy-2-pentenoyl-CoAcan be carried out by a 2-hydroxyacyl-CoA dehydratase. A2-hydroxyglutaryl-CoA dehydratase system has been characterized inAcidaminococcus fermentans and requires both the hgdA and hgdB subunitsand the activator protein, hgdC, for optimal activity (Dutscho et al.,Eur. J. Biochem. 181(3):741-746 (1989); Locher et al. J. Mol. Biol.307(1):297-308; Muller and Buckel, Eur. J. Biochem. 230(2):698-704(2001); Schweiger et al. Eur. J. Biochem. 169(2):441-448 (1987)). Thisenzyme system is similar in mechanism to the lactoyl-CoA dehydratasefrom Clostridium propionicum (Hofmeister and Buckel, Eur. J. Biochem.206(2):547-552 (1992); Kuchta and Abeles, J. Biol. Chem.260(24):13181-13189 (1985)). Homologs to hgdA, hgdB, and hgdC exist inseveral organisms.

Gene GenBank name GI# Accession # Organism hgdA 123112 P11569Acidaminococcus fermentans hgdB 123113 P11570 Acidaminococcus fermentanshgdC 2506909 P11568 Acidaminococcus fermentans hgdA 225177593ZP_03731126.1 Clostridium sp. M62/1 hgdB 225177592 ZP_03731125.1Clostridium sp. M62/1 hgdC 225177594 ZP_03731127.1 Clostridium sp. M62/1hgdA 19703552 NP_603114.1 Fusobacterium nucleatum hgdB 19703553NP_603115.1 Fusobacterium nucleatum hgdC 19703551 NP_603113.1Fusobacterium nucleatum

Conversion of 5-carboxy-2-pentenoyl-CoA to adipate is carried out by theenzymes described in Example I.

The above description provides an exemplary adipate synthesis pathway byway of a 2-hydroxyadipyl-CoA pathway.

Example XI Preparation of an Adipate Producing Microbial Organism Havinga 2-Hydroxyadipyl-CoA Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using a 2-hydroxyadipyl-CoA pathway.

Escherichia coli is used as a target organism to engineer the necessarygenes for adipate synthesis (see FIG. 9). E. coli provides a good hostfor generating a non-naturally occurring microorganism capable ofproducing adipate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleicacids encoding the enzymes utilized in a 2-hydroxyadipyl-CoA to adipatepathway are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel, supra,1999). In particular, the serA (NP_417388.1), gctA (Q59111), and gctB(Q59112)genes encoding the 2-hydroxyadipate dehydrogenase and2-hydroxyadipyl-CoA:acetyl-CoA transferase activities, respectively, arecloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under thePA1/lacO promoter. In addition, the hgdA (P11569), hgdB (P11570), andhgdC (P11568) genes encoding 2-hydroxyadipyl-CoA dehydratase activity,respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. Further, the bcd (NP_349317.1),etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 and AAC73823.1)genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoAsynthetase activities are cloned into a third compatible plasmid, pZS23,under the PA1/lacO promoter. pZS23 is obtained by replacing theampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim,Germany) with a kanamycin resistance module by well-known molecularbiology techniques. The three sets of plasmids are transformed into E.coli strain MG1655 to express the proteins and enzymes required foradipate synthesis.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the2-hydroxyadipyl-CoA pathway genes for adipate synthesis is corroboratedusing methods well known in the art for determining polypeptideexpression or enzymatic activity, including for example, Northern blots,PCR amplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce adipate is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of adipate. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of adipate.Adaptive evolution also can be used to generate better producers of, forexample, the alpha-ketoadipate intermediate or the adipate product.Adaptive evolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the adipate producer to further increaseproduction.

For large-scale production of adipate, the 2-hydroxyadipyl-CoApathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing the culture vessel, forexample, flasks can be sealed with a septum and crimp-cap. Microaerobicconditions also can be utilized by providing a small hole in the septumfor limited aeration. The pH of the medium is maintained at around a pHof 7 by addition of an acid, such as H₂SO₄. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm) and the glucose uptake rate by monitoring carbon source depletionover time. Byproducts such as undesirable alcohols, organic acids, andresidual glucose can be quantified by HPLC (Shimadzu), for example,using an Aminex® series of HPLC columns (for example, HPX-87 series)(BioRad), using a refractive index detector for glucose and alcohols,and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng.775-779 (2005)).

This example describes the preparation of an adipate-producing microbialorganism containing a 2-hydroxyadipyl-CoA pathway.

Example XII Pathways for Production of Hexamethylenediamine, Caprolactamand 6-Aminocaproic Acid

This example describes exemplary pathways for production ofhexamethylenediamine, caprolactam and 6-aminocaproic acid.

Described below are various pathways leading to the production ofcaprolactam, hexamethylenediamine (HMDA), or 6-aminocaproate from commoncentral metabolites. The first described pathway entails the activationof 6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthaseenzyme (FIG. 10, Step Q or R) followed by the spontaneous cyclization of6-aminocaproyl-CoA to form caprolactam (FIG. 10, Step T). The seconddescribed pathway entails the activation of 6-aminocaproate to6-aminocaproyl-CoA (FIG. 10, Step Q or R), followed by a reduction (FIG.10, Step U) and amination (FIG. 10, Step V or W) to form HMDA.6-Aminocaproic acid can alternatively be activated to6-aminocaproyl-phosphate instead of 6-aminocaproyl-CoA.6-Aminocaproyl-phosphate can spontaneously cyclize to form caprolactam.Alternatively, 6-aminocaproyl-phosphate can be reduced to6-aminocaproate semialdehyde, which can be then converted to HMDA asdepicted in FIGS. 10 and 11. In either this case, the amination reactionmust occur relatively quickly to minimize the spontaneous formation ofthe cyclic imine of 6-aminocaproate semialdehyde. Linking or scaffoldingthe participating enzymes represents a potentially powerful option forensuring that the 6-aminocaproate semialdehyde intermediate isefficiently channeled from the reductase enzyme to the amination enzyme.

Another option for minimizing or even eliminating the formation of thecyclic imine or caprolactam during the conversion of 6-aminocaproic acidto HMDA entails adding a functional group (for example, acetyl,succinyl) to the amine group of 6-aminocaproic acid to protect it fromcyclization. This is analogous to ornithine formation from L-glutamatein Escherichia coli. Specifically, glutamate is first converted toN-acetyl-L-glutamate by N-acetylglutamate synthase. N-Acetyl-L-glutamateis then activated to N-acetylglutamylphosphate, which is reduced andtransaminated to form N-acetyl-L-ornithine. The acetyl group is thenremoved from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylaseforming L-ornithine. Such a route is necessary because formation ofglutamate-5-phosphate from glutamate followed by reduction toglutamate-5-semialdehyde leads to the formation of(S)-1-pyrroline-5-carboxylate, a cyclic imine formed spontaneously fromglutamate-5-semialdehyde. In the case of forming HMDA from6-aminocaproic acid, the steps can involve acetylating 6-aminocaproicacid to acetyl-6-aminocaproic acid, activating the carboxylic acid groupwith a CoA or phosphate group, reducing, aminating, and deacetylating.

Note that 6-aminocaproate can be formed from various starting molecules.For example, the carbon backbone of 6-aminocaproate can be derived fromsuccinyl-CoA and acetyl-CoA as depicted in FIG. 10 and also described inFIGS. 2, 3 and 8. Alternatively, 6-aminocaproate can be derived fromalpha-ketoadipate, where alpha-ketoadipate is converted to adipyl-CoA(see FIG. 9), and adipyl-CoA is converted to 6-aminocaproate as shown inFIG. 10.

FIG. 11 provides two additional metabolic pathways to 6-aminocaproate or6-aminocapropyl-CoA starting from 4-aminobutyryl-CoA and acetyl-CoA. Thefirst route entails the condensation of 4-aminobutyryl-CoA andacetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (Step A) followed by areduction (Step B), dehydration (Step C), and reduction (Step D) to form6-aminocaproyl-CoA. 6-Aminocaproyl-CoA can be converted to6-aminocaproate by a transferase (Step K), synthase (Step L), orhydrolase (Step M) enzyme. Alternatively, 6-aminocaproyl-CoA can beconverted to caprolactam by spontaneous cyclization (Step Q) or to HMDAfollowing its reduction (Step N) and amination (Step O or P). The secondpathway described in FIG. 11 entails the condensation of4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA(Step A) which is then converted to 3-oxo-6-aminohexanoate by atransferase (Step E), synthase (Step F), or hydrolase (Step G).3-Oxo-6-aminohexanoate is then reduced (Step H), dehydrated (Step I),and reduced (Step J) to form 6-aminocaproate.

The starting molecule, 4-aminobutyryl-CoA, can be formed from variouscommon central metabolites. For example, glutamate can be decarboxylatedto 4-aminobutyrate, which is then activated by a CoA-transferase orsynthase to 4-aminobutyryl-CoA. Alternatively, succinate semialdehyde,formed from either the reduction of succinyl-CoA or the decarboxylationof alpha-ketoglutarate, can be transaminated to 4-aminobutyrate prior toactivation by a CoA-transferase or synthase to form 4-aminobutyryl-CoA.It is noted that 4-aminobutyryl-CoA and several of the intermediates ofthe 4-aminobutyryl-CoA to 6-aminocaproyl-CoA pathway may spontaneouslycyclize to their corresponding lactams. Thus, adding a protectivefunctional group to the terminal amine group of 4-aminobutyryl-CoAand/or several of the amino-CoA intermediates can be used to minimizethe formation of unwanted cyclic byproducts. In this case, the samegeneral set of transformations depicted in FIG. 11 would apply, althoughtwo additional steps, for example, an acetylase and deacetylase, can beadded to the pathway.

All transformations depicted in FIGS. 10-11 fall into the 12 generalcategories of transformations shown in Table 8. Below is described anumber of biochemically characterized candidate genes in each category.Specifically listed are genes that can be applied to catalyze theappropriate transformations in FIGS. 10-11 when cloned and expressed.

TABLE 8 Enzyme types for conversion of succinyl-CoA, acetyl-CoA, and/or4-aminobutyryl-CoA to 6-aminocaproate, caprolactam, and/orhexamethylenediamine. The first three digits of each label correspond tothe first three Enzyme Commission number digits which denote the generaltype of transformation independent of substrate specificity. LabelFunction 1.1.1.a Oxidoreductase (ketone to hydroxyl or aldehyde toalcohol) 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.3.1.aOxidoreductase operating on CH—CH donors 1.4.1.a Oxidoreductaseoperating on amino acids 2.3.1.b Acyltransferase 2.6.1.aAminotransferase 2.8.3.a Coenzyme-A transferase 3.1.2.a Thiolesterhydrolase (CoA specific) 4.2.1.a Hydro-lyase 6.2.1.a Acid-thiol ligase6.3.1.a/ Amide synthases/peptide synthases 6.3.2.a No enzyme Spontaneouscyclization required

1.1.1.a Oxidoreductases.

Four transformations depicted in FIGS. 10 and 11 require oxidoreductasesthat convert a ketone functionality to a hydroxyl group. Step B in bothFIGS. 10 and 11 involves converting a 3-oxoacyl-CoA to a3-hydroxyacyl-CoA. Step H in both FIGS. 1 and 2 involves converting a3-oxoacid to a 3-hydroxyacid.

Exemplary enzymes that can convert 3-oxoacyl-CoA molecules such as3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA into 3-hydroxyacyl-CoAmolecules such as 3-hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA,respectively, include enzymes whose natural physiological roles are infatty acid beta-oxidation or phenylacetate catabolism. For example,subunits of two fatty acid oxidation complexes in E. coli, encoded byfadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock etal., Methods Enzymol. 71:403-411 (1981)). Furthermore, the gene productsencoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl.Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescensST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) catalyze thereverse reaction of step B in FIG. 10, that is, the oxidation of3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism ofphenylacetate or styrene. Note that the reactions catalyzed by suchenzymes are reversible. In addition, given the proximity in E. coli ofpaaH to other genes in the phenylacetate degradation operon (Nogales etal., Microbiology 153:357-365 (2007)) and the fact that paaH mutantscannot grow on phenylacetate (Ismail et al., Eur. J Biochem.270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodesa 3-hydroxyacyl-CoA dehydrogenase.

GenBank Gene name GI# Accession # Organism fadB 119811 P21177.2Escherichia coli fadJ 3334437 P77399.1 Escherichia coli paaH 16129356NP_415913.1 Escherichia coli phaC 26990000 NP_745425.1 Pseudomonasputida paaC 106636095 ABF82235.1 Pseudomonas fluorescens

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoAmolecules to their corresponding 3-hydroxyacyl-CoA molecules include3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridiumacetobutylicum, encoded by hbd, has been cloned and functionallyexpressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807(1989)). Additional gene candidates include Hbd1 (C-terminal domain) andHbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer et al., FEBSLett. 21:351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al., JBiol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstratedto reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloearamigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)) and phaBfrom Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61:297-309(2006)). The former gene candidate is NADPH-dependent, its nucleotidesequence has been determined (Peoples et al., Mol. Microbiol 3:349-357(1989)) and the gene has been expressed in E. coli. Substratespecificity studies on the gene led to the conclusion that it couldaccept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Plouxet al., supra).

GenBank Gene name GI# Accession # Organism hbd 18266893 P52041.2Clostridium acetobutylicum Hbd2 146348271 EDK34807.1 Clostridiumkluyveri Hbd1 146345976 EDK32512.1 Clostridium kluyveri HSD17B10 3183024O02691.3 Bos taurus phbB 130017 P23238.1 Zoogloea ramigera phaB146278501 YP_001168660.1 Rhodobacter sphaeroides

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)).

GenBank Gene name GI# Accession # Organism hbd 15895965 NP_349314.1Clostridium acetobutylicum hbd 20162442 AAM14586.1 Clostridiumbeijerinckii Msed_1423 146304189 YP_001191505 Metallosphaera sedulaMsed_0399 146303184 YP_001190500 Metallosphaera sedula Msed_0389146303174 YP_001190490 Metallosphaera sedula Msed_1993 146304741YP_001192057 Metallosphaera sedula

Various alcohol dehydrogenases represent good candidates for converting3-oxoadipate to 3-hydroxyadipate (step H, FIG. 10) or3-oxo-6-aminohexanoate to 3-hydroxy-6-aminohexanoate (step H, FIG. 11).Two such enzymes capable of converting an oxoacid to a hydroxyacid areencoded by the malate dehydrogenase (mdh) and lactate dehydrogenase(ldhA) genes in E. coli. In addition, lactate dehydrogenase fromRalstonia eutropha has been shown to demonstrate high activities onsubstrates of various chain lengths such as lactate, 2-oxobutyrate,2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem.130:329-334 (1983)). Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.,Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys. Res. Commun. 77:586-591 (1977)). An additional candidate forthese steps is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh)from the human heart which has been cloned and characterized (Marks etal., J Biol. Chem. 267:15459-15463 (1992)). This enzyme is adehydrogenase that operates on a 3-hydroxyacid. Another exemplaryalcohol dehydrogenase converts acetone to isopropanol as was shown in C.beijerinckii (Ismaiel et al., J Bacteriol. 175:5097-5105 (1993) and T.brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al.,Biochemistry 28:6549-6555 (1989)).

Gene GenBank name GI# Accession # Organism mdh 1789632 AAC76268.1Escherichia coli ldhA 16129341 NP_415898.1 Escherichia coli ldh113866693 YP_725182.1 Ralstonia eutropha bdh 177198 AAA58352.1 Homosapiens adh 60592974 AAA23199.2 Clostridium beijerinckii adh 113443P14941.1 Thermoanaerobacter brockii

1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde).

The transformations of adipyl-CoA to adipate semialdehyde (Step N, FIG.10) and 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde (Step U, FIG.10; Step N, FIG. 11) require acyl-CoA dehydrogenases capable of reducingan acyl-CoA to its corresponding aldehyde. Exemplary genes that encodesuch enzymes include the Acinetobacter calcoaceticus acr1 encoding afatty acyl-CoA reductase (Reiser et al., J. Bacteriology 179:2969-2975(1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige etal., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- andNADP-dependent succinate semialdehyde dehydrogenase encoded by the sucDgene in Clostridium kluyveri (Sohling et al., J. Bacteriol. 178:871-880(1996)). SucD of P. gingivalis is another succinate semialdehydedehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)).The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp,encoded by bphG, is yet another candidate as it has been demonstrated tooxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde,isobutyraldehyde and formaldehyde (Powlowski et al., J Bacteriol.175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, theenzyme encoded by adhE in Leuconostoc mesenteroides has been shown tooxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA(Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al.,Biotechnol Lett. 27:505-510 (2005)).

GenBank Gene name GI# Accession # Organism acr1 50086359 YP_047869.1Acinetobacter calcoaceticus acr1 1684886 AAC45217 Acinetobacter baylyiacr1 18857901 BAB85476.1 Acinetobacter sp. Strain M-1 sucD 172046062P38947.1 Clostridium kluyveri sucD 34540484 NP_904963.1 Porphyromonasgingivalis bphG 425213 BAA03892.1 Pseudomonas sp adhE 55818563AAV66076.1 Leuconostoc mesenteroides

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., supra; Thauer R. K.,Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactorand has been characterized in Metallosphaera and Sulfolobus spp (Alberet al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol.184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 inMetallosphaera sedula (Alber et al., supra; Berg et al., supra). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., supra). This enzymehas also been shown to catalyze the conversion of methylmalonyl-CoA toits corresponding aldehyde (WO/2007/141208). Although the aldehydedehydrogenase functionality of these enzymes is similar to thebifunctional dehydrogenase from Chloroflexus aurantiacus, there islittle sequence similarity. Both malonyl-CoA reductase enzyme candidateshave high sequence similarity to aspartate-semialdehyde dehydrogenase,an enzyme catalyzing the reduction and concurrent dephosphorylation ofaspartyl-4-phosphate to aspartate semialdehyde. Additional genecandidates can be found by sequence homology to proteins in otherorganisms including Sulfolobus solfataricus and Sulfolobusacidocaldarius and have been listed below. Yet another candidate forCoA-acylating aldehyde dehydrogenase is the ald gene from Clostridiumbeijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)).This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA totheir corresponding aldehydes. This gene is very similar to eutE thatencodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli(Toth et al., supra).

GenBank Gene name GI# Accession # Organism Msed_0709 146303492ΥP_001190808.1 Metallosphaera sedula mcr 15922498 NP_378167.1 Sulfolobustokodaii asd-2 15898958 NP_343563.1 Sulfolobus solfataricus Saci_237070608071 YP_256941.1 Sulfolobus acidocaldarius Ald 49473535 AAT66436Clostridium beijerinckii eutE 687645 AAA80209 Salmonella typhimuriumeutE 2498347 P77445 Escherichia coli

1.3.1.a Oxidoreductase Operating on CH—CH Donors.

Referring to FIG. 10, step D refers to the conversion of5-carboxy-2-pentenoyl-CoA to adipyl-CoA by 5-carboxy-2-pentenoyl-CoAreductase. Referring to FIG. 11, step D refers to the conversion of6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA. Enoyl-CoA reductaseenzymes are suitable enzymes for either transformation. One exemplaryenoyl-CoA reductase is the gene product of bcd from C. acetobutylicum(Boynton et al., J Bacteriol. 178:3015-3024 (1996); Atsumi et al.,Metab. Eng. 2008 10(6):305-311 (2008)(Epub Sep. 14, 2007), whichnaturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA.Activity of this enzyme can be enhanced by expressing bcd in conjunctionwith expression of the C. acetobutylicum etfAB genes, which encode anelectron transfer flavoprotein. An additional candidate for theenoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase fromE. gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). Aconstruct derived from this sequence following the removal of itsmitochondrial targeting leader sequence was cloned in E. coli resultingin an active enzyme (Hoffmeister et al., supra). This approach is wellknown to those skilled in the art of expressing eukaryotic genes,particularly those with leader sequences that may target the geneproduct to a specific intracellular compartment, in prokaryoticorganisms. A close homolog of this gene, TDE0597, from the prokaryoteTreponema denticola represents a third enoyl-CoA reductase which hasbeen cloned and expressed in E. coli (Tucci et al., FEBS Letters581:1561-1566 (2007)).

GenBank Gene name GI# Accession # Organism bcd 15895968 NP_349317.1Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridiumacetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum TER62287512 Q5EU90.1 Euglena gracilis TDE0597 42526113 NP_971211.1Treponema denticola

Step J of both FIGS. 10 and 11 requires a 2-enoate reductase enzyme.2-Enoate reductases (EC 1.3.1.31) are known to catalyze theNAD(P)H-dependent reduction of a wide variety of α, β-unsaturatedcarboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem.276:5779-5787 (2001)). 2-Enoate reductase is encoded by enr in severalspecies of Clostridia (Giesel et al., Arch Microbiol 135:51-57 (1983))including C. tyrobutyricum, and C. thermoaceticum (now called Moorellathermoaceticum) (Rohdich et al., supra). In the published genomesequence of C. kluyveri, 9 coding sequences for enoate reductases havebeen reported, out of which one has been characterized (Seedorf et al.,Proc. Natl. Acad. Sci. USA, 105:2128-2133 (2008)). The enr genes fromboth C. tyrobutyricum and C. thermoaceticum have been cloned andsequenced and show 59% identity to each other. The former gene is alsofound to have approximately 75% similarity to the characterized gene inC. kluyveri (Giesel et al., supra). It has been reported based on thesesequence results that enr is very similar to the dienoyl CoA reductasein E. coli (fadH) (Rohdich et al., supra). The C. thermoaceticum enrgene has also been expressed in an enzymatically active form in E. coli(Rohdich et al., supra).

GenBank Gene name GI# Accession # Organism fadH 16130976 NP_417552.1Escherichia coli enr 169405742 ACA54153.1 Clostridium botulinum A3 strenr 2765041 CAA71086.1 Clostridium tyrobutyricum enr 3402834 CAA76083.1Clostridium kluyveri enr 83590886 YP_430895.1 Moorella thermoacetica

1.4.1.a Oxidoreductase Operating on Amino Acids.

FIG. 10 depicts two reductive aminations. Specifically, step P of FIG.10 involves the conversion of adipate semialdehyde to 6-aminocaproateand step W of FIG. 10 entails the conversion of 6-aminocaproatesemialdehyde to hexamethylenediamine. The latter transformation is alsorequired in FIG. 11, Step P.

Most oxidoreductases operating on amino acids catalyze the oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor, thoughthe reactions are typically reversible. Exemplary oxidoreductasesoperating on amino acids include glutamate dehydrogenase (deaminating),encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh,and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhAgene product from Escherichia coli (McPherson et al., Nucleic. AcidsRes. 11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273(1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998);Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 fromHalobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005))catalyze the reversible interconversion of glutamate to 2-oxoglutarateand ammonia, while favoring NADP(H), NAD(H), or both, respectively. Theldh gene of Bacillus cereus encodes the LeuDH protein that has a wide ofrange of substrates including leucine, isoleucine, valine, and2-aminobutanoate (Stoyan et al., J. Biotechnol 54:77-80 (1997); Ansorgeet al., Biotechnol Bioeng. 68:557-562 (2000)). The nadX gene fromThermotoga maritime encoding for the aspartate dehydrogenase is involvedin the biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808(2003)).

GenBank Gene name GI# Accession # Organism gdhA 118547 P00370Escherichia coli gdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827NP_279651.1 Halobacterium salinarum ldh 61222614 P0A393 Bacillus cereusnadX 15644391 NP_229443.1 Thermotoga maritima

The lysine 6-dehydrogenase (deaminating), encoded by the lysDH genes,catalyze the oxidative deamination of the ε-amino group of L-lysine toform 2-aminoadipate-6-semialdehyde, which in turn nonenzymaticallycyclizes to form Δ¹-piperideine-6-carboxylate (Misono et al., J.Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found inGeobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., JBiochem 106:76-80 (1989); Misono et al., supra), and Achromobacterdenitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)).Such enzymes are particularly good candidates for converting adipatesemialdehyde to 6-aminocaproate given the structural similarity betweenadipate semialdehyde and 2-aminoadipate-6-semialdehyde.

GenBank Gene name GI# Accession # Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

2.3.1.b Acyl Transferase.

Referring to FIG. 10, step A involves 3-oxoadipyl-CoA thiolase, orequivalently, succinyl CoA:acetyl CoA acyl transferase (β-ketothiolase).The gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabeket al., J. Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U(Olivera et al., supra), paaE in Pseudomonas fluorescens ST (Di Gennaroet al., supra), and paaJ from E. coli (Nogales et al., supra) catalyzethe conversion of 3-oxoadipyl-CoA into succinyl-CoA and acetyl-CoAduring the degradation of aromatic compounds such as phenylacetate orstyrene. Since β-ketothiolase enzymes catalyze reversibletransformations, these enzymes can be employed for the synthesis of3-oxoadipyl-CoA. For example, the ketothiolase phaA from R. eutrophacombines two molecules of acetyl-CoA to form acetoacetyl-CoA (Sato etal., J Biosci Bioeng 103:38-44 (2007)). Similarly, a β-keto thiolase(bktB) has been reported to catalyze the condensation of acetyl-CoA andpropionyl-CoA to form β-ketovaleryl-CoA (Slater et al., J. Bacteriol.180:1979-1987 (1998)) in R. eutropha. In addition to the likelihood ofpossessing 3-oxoadipyl-CoA thiolase activity, all such enzymes representgood candidates for condensing 4-aminobutyryl-CoA and acetyl-CoA to form3-oxo-6-aminohexanoyl-CoA (step A, FIG. 11) either in their native formsor once they have been appropriately engineered.

GenBank Gene name GI# Accession # Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida paaE 106636097 ABF82237.1Pseudomonas fluorescens

2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase (AKPT) enzymespresent additional candidates for performing step A in FIGS. 10 and 11.AKPT is a pyridoxal phosphate-dependent enzyme participating inornithine degradation in Clostridium sticklandii (Jeng et al.,Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology145:819-826 (1999)). A gene cluster encoding the alpha and beta subunitsof AKPT (or-2 (ortA) and or-3 (ortB)) was recently identified and thebiochemical properties of the enzyme were characterized (Fonknechten etal., J. Bacteriol. In Press (2009)). The enzyme is capable of operatingin both directions and naturally reacts with the D-isomer of alanine.AKPT from Clostridium sticklandii has been characterized but its proteinsequence has not yet been published. Enzymes with high sequence homologyare found in Clostridium difficile, Alkaliphilus metalliredigenes QYF,Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4(Fonknechten et al., supra).

GenBank Gene name GI# Accession # Organism ortA (α) 126698017YP_001086914.1 Clostridium difficile 630 ortB (β) 126698018YP_001086915.1 Clostridium difficile 630 Amet_2368 (α) 150390132YP_001320181.1 Alkaliphilus metalliredigenes QYF Amet_2369 (β) 150390133YP_001320182.1 Alkaliphilus metalliredigenes QYF Teth514_1478 (α)167040116 YP_001663101.1 Thermoanaerobacter sp. X514 Teth514_1479 (β)167040117 YP_001663102.1 Thermoanaerobacter sp. X514 TTE1235 (α)20807687 NP_622858.1 Thermoanaerobacter tengcongensis MB4 thrC (β)20807688 NP_622859.1 Thermoanaerobacter tengcongensis MB4

2.6.1.a Aminotransferase.

Step O of FIGS. 10 and 11 and Step V of FIG. 10 require transaminationof a 6-aldehyde to an amine. These transformations can be catalyzed bygamma-aminobutyrate transaminase (GABA transaminase). One E. coli GABAtransaminase is encoded by gabT and transfers an amino group fromglutamate to the terminal aldehyde of succinyl semialdehyde (Bartsch etal., J. Bacteriol. 172:7035-7042 (1990)). The gene product of puuEcatalyzes another 4-aminobutyrate transaminase in E. coli (Kurihara etal., J. Biol. Chem. 280:4602-4608 (2005)). GABA transaminases in Musmusculus, Pseudomonas fluorescens, and Sus scrofa have been shown toreact with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82(1985); Scott et al., J. Biol. Chem. 234:932-936 (1959)).

GenBank Gene name GI# Accession # Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. Such enzymes are particularly wellsuited for carrying out the conversion of 6-aminocaproate semialdehydeto hexamethylenediamine. The E. coli putrescine aminotransferase isencoded by the ygjG gene and the purified enzyme also was able totransaminate cadaverine and spermidine (Samsonova et al., BMC Microbiol3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptaneand with amino acceptors other than 2-oxoglutarate (e.g., pyruvate,2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, K. H.,J Biol Chem 239:783-786 (1964)). A putrescine aminotransferase withhigher activity with pyruvate as the amino acceptor thanalpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu etal., J Bacteriol 184:3765-3773 (2002)).

GenBank Gene name GI# Accession # Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa

Yet additional candidate enzymes includebeta-alanine/alpha-ketoglutarate aminotransferases which producemalonate semialdehyde from beta-alanine (WO08027742). The gene productof SkPYD4 in Saccharomyces kluyveri was also shown to preferentially usebeta-alanine as the amino group donor (Andersen et al., FEBS. J.274:1804-1817 (2007)). SkUGA1 encodes a homologue of Saccharomycescerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem.,149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in bothβ-alanine and GABA transamination (Andersen et al., supra).3-Amino-2-methylpropionate transaminase catalyzes the transformationfrom methylmalonate semialdehyde to 3-amino-2-methylpropionate. Thisenzyme has been characterized in Rattus norvegicus and Sus scrofa and isencoded by Abat (Tamaki et al, Methods Enzymol, 324:376-389 (2000)).

GenBank Gene name GI# Accession # Organism SkyPYD4 98626772 ABF58893.1Saccharomyces kluyveri SkUGA1 98626792 ABF58894.1 Saccharomyces kluyveriUGA1 6321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191P50554.3 Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

2.8.3.a Coenzyme-A Transferase.

CoA transferases catalyze reversible reactions that involve the transferof a CoA moiety from one molecule to another. For example, step E ofFIG. 10 is catalyzed by a 3-oxoadipyl-CoA transferase. In this step,3-oxoadipate is formed by the transfer of the CoA group from3-oxoadipyl-CoA to succinate, acetate, or another CoA acceptor. Step Eof FIG. 11 entails the transfer of a CoA moiety from another3-oxoacyl-CoA, 3-oxo-6-aminohexanoyl-CoA. One candidate enzyme for thesesteps is the two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas,which has been shown to have 3-oxoadipyl-CoA/succinate transferaseactivity (Kaschabek et al., supra). Similar enzymes based on homologyexist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30(1994)) and Streptomyces coelicolor. Additional exemplarysuccinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997))and Bacillus subtilis (Stols et al., Protein. Expr. Purif. 53:396-403(2007)).

GenBank Gene name GI# Accession # Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida peaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 peaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

A 3-oxoacyl-CoA transferase that can utilize acetate as the CoA acceptoris acetoacetyl-CoA transferase, encoded by the E. coli atoA (alphasubunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys. Res Commun. 33:902-908 (1968); Korolev et al., ActaCrystallogr. D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme hasalso been shown to transfer the CoA moiety to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthieset al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate(Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra).Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncanet al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridiumacetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583(1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci. Biotechnol Biochem. 71:58-68 (2007)).

GenBank Gene name GI# Accession # Organism atoA 2492994 P76459.1Escherichia coli K12 atoD 2492990 P76458.1 Escherichia coli K12 actA62391407 YP_226809.1 Corynebacterium glutamicum ATCC 13032 cg059262389399 YP_224801.1 Corynebacterium glutamicum ATCC 13032 ctfA 15004866NP_149326.1 Clostridium acetobutylicum ctfB 15004867 NP_149327.1Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridiumsaccharoperbutylacetonicum

The above enzymes may also exhibit the desired activities on adipyl-CoAand adipate (FIG. 10, step K) or 6-aminocaproate and 6-aminocaproyl-CoA(FIG. 10, step Q; FIG. 2, step K). Nevertheless, additional exemplarytransferase candidates are catalyzed by the gene products of cat1, cat2,and cat3 of Clostridium kluyveri which have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferaseactivity, respectively (Seedorf et al., supra; Sohling et al., Eur. JBiochem. 212:121-127 (1993); Sohling et al., J Bacteriol. 178:871-880(1996)).

GenBank Gene name GI# Accession # Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3146349050 EDK35586.1 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

GenBank Gene name GI# Accession # Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

3.1.2.a Thiolester Hydrolase (CoA Specific).

Several eukaryotic acetyl-CoA hydrolases have broad substratespecificity and thus represent suitable candidate enzymes forhydrolyzing 3-oxoadipyl-CoA, adipyl-CoA, 3-oxo-6-aminohexanoyl-CoA, or6-aminocaproyl-CoA (Steps G and M of FIGS. 10 and 11). For example, theenzyme from Rattus norvegicus brain (Robinson et al., Biochem. Biophys.Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoAand malonyl-CoA.

GenBank Gene name GI# Accession # Organism acot12 18543355 NP_570103.1Rattus norvegicus

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra; Shimomura et al., Methods Enzymol. 324:229-240 (2000))and Homo sapiens (Shimomura et al., supra). Candidate genes by sequencehomology include hibch of Saccharomyces cerevisiae and BC 2292 ofBacillus cereus.

GenBank Gene name GI# Accession # Organism hibch 146324906 Q5XIE6.2Rattus norvegicus hibch 146324905 Q6NVY1.2 Homo sapiens hibch 2506374P28817.2 Saccharomyces cerevisiae BC_2292 29895975 AP09256 Bacilluscereus

Yet another candidate hydrolase is the human dicarboxylic acidthioesterase, acot8, which exhibits activity on glutaryl-CoA,adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin etal., J. Biol. Chem. 280:38125-38132 (2005)) and the closest E. colihomolog, tesB, which can also hydrolyze a broad range of CoA thiolesters(Naggert et al., J. Biol Chem 266:11044-11050 (1991)). A similar enzymehas also been characterized in the rat liver (Deana R., Biochem Int26:767-773 (1992)).

Gene name GI# GenBank Accession # Organism tesB 16128437 NP_414986Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669NP_570112 Rattus norvegicus

Other potential E. coli thiolester hydrolases include the gene productsof tesA (Bonner et al., J Biol Chem 247:3123-3133 (1972)), ybgC(Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); Zhuang et al.,FEBS Lett 516:161-163 (2002)), paaI (Song et al., J Biol Chem281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol189:7112-7126 (2007)).

Gene name GI# GenBank Accession # Organism tesA 16128478 NP_415027Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paaI 16129357NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli

6.3.1.a/6.3.2.a Amide Synthases/Peptide Synthases.

The direct conversion of 6-aminocaproate to caprolactam (Step S, FIG.10; Step R, FIG. 11) requires the formation of an intramolecular peptidebond. Ribosomes, which assemble amino acids into proteins duringtranslation, are nature's most abundant peptide bond-forming catalysts.Nonribosomal peptide synthetases are peptide bond forming catalysts thatdo not involve messenger mRNA (Schwarzer et al., Nat Prod. Rep.20:275-287 (2003)). Additional enzymes capable of forming peptide bondsinclude acyl-CoA synthetase from Pseudomonas chlororaphis (Abe et al., JBiol Chem 283:11312-11321 (2008)), gamma-Glutamylputrescine synthetasefrom E. coli (Kurihara et al., J Biol Chem 283:19981-19990 (2008)), andbeta-lactam synthetase from Streptomyces clavuligerus (Bachmann et al.,Proc Natl Acad Sci USA 95:9082-9086 (1998); Bachmann et al.,Biochemistry 39:11187-11193 (2000); Miller et al., Nat Struct. Biol8:684-689 (2001); Miller et al., Proc Natl Acad Sci USA 99:14752-14757(2002); Tahlan et al., Antimicrob. Agents. Chemother. 48:930-939(2004)).

GenBank Gene name GI# Accession # Organism acsA 60650089 BAD90933Pseudomonas chlororaphis puuA 87081870 AAC74379 Escherichia coli bls41016784 Q9R8E3 Streptomyces clavuligerus

4.2.1.a Hydrolyase.

Most dehydratases catalyze the α, β-elimination of water. This involvesactivation of the α-hydrogen by an electron-withdrawing carbonyl,carboxylate, or CoA-thiol ester group and removal of the hydroxyl groupfrom the β-position. Enzymes exhibiting activity on substrates with anelectron-withdrawing carboxylate group are excellent candidates fordehydrating 3-hydroxyadipate (FIG. 10, Step I) or3-hydroxy-6-aminohexanoate (FIG. 11, Step I).

For example, fumarase enzymes naturally catalyze the reversibledehydration of malate to fumarate. E. coli has three fumarases: FumA,FumB, and FumC that are regulated by growth conditions. FumB is oxygensensitive and only active under anaerobic conditions. FumA is activeunder microanaerobic conditions, and FumC is the only active enzyme inaerobic growth (Tseng et al., J Bacteriol 183:461-467 (2001); Woods etal., Biochim Biophys Acta 954:14-26 (1988); Guest et al., J GenMicrobiol 131:2971-2984 (1985)). Additional enzyme candidates are foundin Campylobacter jejuni (Smith et al., Int. J Biochem. Cell Biol31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi etal., J Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequencehomology include fum1 from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum. The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

Gene GenBank name GI# Accession # Organism fumA 81175318 P0AC33Escherichia coli fumB 33112655 P14407 Escherichia coli fumC 120601P05042 Escherichia coli fumC 9789756 O69294 Campylobacter jejuni fumC3062847 BAA25700 Thermus thermophilus fumH 120605 P14408 Rattusnorvegicus fum1 39931311 P93033 Arabidopsis thaliana fumC 39931596Q8NRN8 Corynebacterium glutamicum MmcB 147677691 YP_001211906Pelotomaculum thermopropionicum MmcC 147677692 YP_001211907Pelotomaculum thermopropionicum

Two additional dehydratase candidates are 2-(hydroxymethyl)glutaratedehydratase and dimethylmaleate hydratase, enzymes studied for theirrole in nicontinate catabolism in Eubacterium barkeri (formerlyClostridium barkeri) (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6(2006)). 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containingenzyme that dehydrates 2-(hydroxymethyl)glutarate to2-methylene-glutarate. This enzyme is encoded by hmd in Eubacteriumbarkeri (Alhapel et al., supra). Similar enzymes with high sequencehomology are found in Bacteroides capillosus, Anaerotruncus colihominis,and Natranaerobius thermophilius. These enzymes are homologous to thealpha and beta subunits of [4Fe-4S]-containing bacterial serinedehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA).

GenBank Gene name GI# Accession # Organism hmd 86278275 ABC88407.1Eubacterium barkeri BACCAP_02294 154498305 ZP_02036683.1 Bacteroidescapillosus ANACOL_02527 167771169 ZP_02443222.1 Anaerotruncuscolihominis DSM 17241 NtherDRAFT_2368 169192667 ZP_02852366.1Natranaerobius thermophilus JW/NM-WN-LF

Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe²⁺-dependentand oxygen-sensitive enzyme in the aconitase family that hydratesdimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme isencoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra;Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857(1984)).

Gene name GI# GenBank Accession # Organism dmdA 86278276 ABC88408Eubacterium barkeri dmdB 86278277 ABC88409.1 Eubacterium barkeri

An additional enzyme candidate is 2-methylmalate dehydratase, alsocalled citramalate hydrolyase, a reversible hydrolyase that catalyzesthe alpha, beta elimination of water from citramalate to formmesaconate. This enzyme has been purified and characterized inClostridium tetanomorphum (Wang et al., J Biol. Chem. 244:2516-2526(1969)). The activity of this enzyme has also been detected in severalbacteria in the genera Citrobacter and Morganella in the context of theglutamate degradation VI pathway (Kato et al., Arch. Microbiol168:457-463 (1997)). Genes encoding this enzyme have not been identifiedin any organism to date.

Enzymes exhibiting activity on substrates with an electron-withdrawingCoA-thiol ester group adjacent to the α-hydrogen are excellentcandidates for dehydrating 3-hydroxyadipyl-CoA (FIG. 10, Step C) or3-hydroxy-6-aminohexanoyl-CoA (FIG. 11, Step C). The enoyl-CoAhydratases, phaA and phaB, of P. putida are believed to carry out thehydroxylation of double bonds during phenylacetate catabolism (Oliveraet al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). The paaA andpaaB from P. fluorescens catalyze analogous transformations (Olivera etal., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). Lastly, a numberof Escherichia coli genes have been shown to demonstrate enoyl-CoAhydratase functionality including maoC (Park et al., J Bacteriol.185:5391-5397 (2003)), paaF (Ismail et al., supra; Park et al., Appl.Biochem. Biotechnol 113-116:335-346 (2004); Park et al., BiotechnolBioeng 86:681-686 (2004)) and paaG (Ismail et al., supra; Park et al.,Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al.,Biotechnol Bioeng 86:681-686 (2004)). Crotonase enzymes are additionalcandidates for dehydrating the required 3-hydroxyacyl-CoA moleculesdepicted in FIGS. 10 and 11. These enzymes are required for n-butanolformation in some organisms, particularly Clostridial species, and alsocomprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle inthermoacidophilic Archaea of the genera Sulfolobus, Acidianus, andMetallosphaera. Exemplary genes encoding crotonase enzymes can be foundin C. acetobutylicum (Boynton et al., supra), C. kluyveri (Hillmer etal., FEBS Lett. 21:351-354 (1972)), and Metallosphaera sedula (Berg etal., supra) though the sequence of the latter gene is not known.Enoyl-CoA hydratases, which are involved in fatty acid beta-oxidationand/or the metabolism of various amino acids, can also catalyze thehydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg. Med. Chem.11:9-20 (2003); Conrad et al., J Bacteriol. 118:103-111 (1974)).

Gene GenBank name GI# Accession # Organism paaA 26990002 NP_745427.1Pseudomonas fluorescens paaB 26990001 NP_745426.1 Pseudomonasfluorescens phaA 106636093 ABF82233.1 Pseudomonas putida phaB 106636094ABF82234.1 Pseudomonas putida maoC 16129348 NP_415905.1 Escherichia colipaaF 16129354 NP_415911.1 Escherichia coli paaG 16129355 NP_415912.1Escherichia coli crt 15895969 NP_349318.1 Clostridium acetobutylicumcrt1 153953091 YP_001393856 Clostridium kluyveri DSM 555

6.2.1.a Acid-Thiol Ligase.

Steps F, L, and R of FIG. 10 and Steps F and L of FIG. 11 requireacid-thiol ligase or synthetase functionality (the terms ligase,synthetase, and synthase are used herein interchangeably and refer tothe same enzyme class). Exemplary genes encoding enzymes likely to carryout these transformations include the sucCD genes of E. coli whichnaturally form a succinyl-CoA synthetase complex. This enzyme complexnaturally catalyzes the formation of succinyl-CoA from succinate withthe concaminant consumption of one ATP, a reaction which is reversiblein vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Given thestructural similarity between succinate and adipate, that is, both arestraight chain dicarboxylic acids, it is reasonable to expect someactivity of the sucCD enzyme on adipyl-CoA.

Gene name GI# GenBank Accession # Organism sucC 16128703 NP_415256.1Escherichia coli sucD 1786949 AAC73823.1 Escherichia coli

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical Journal 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower etal., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgamiet al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyzethe ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.

Gene name GI# GenBank Accession # Organism phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida bioW 50812281NP_390902.2 Bacillus subtilis AACS 21313520 NP_084486.1 Mus musculusAACS 31982927 NP_076417.2 Homo sapiens

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch Microbiol182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). Theenzymes from A. fulgidus, H. marismortui and P. aerophilum have all beencloned, functionally expressed, and characterized in E. coli (Musfeldtet al., supra; Brasen et al., supra).

GenBank Gene name GI# Accession # Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 scs 55377722 YP_135572.1 Haloarculamarismortui ATCC 43049 PAE3250 18313937 NP_560604.1 Pyrobaculumaerophilum str. IM2

Yet another option is to employ a set of enzymes with net ligase orsynthetase activity. For example, phosphotransadipylase and adipatekinase enzymes are catalyzed by the gene products of buk1, buk2, and ptbfrom C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang etal., J Mol. Microbiol. Biotechnol. 2:33-38 (2000)). The ptb gene encodesan enzyme that can convert butyryl-CoA into butyryl-phosphate, which isthen converted to butyrate via either of the buk gene products with theconcomitant generation of ATP.

GenBank Gene name GI# Accession # Organism ptb 15896327 NP_349676Clostridium acetobutylicum buk1 15896326 NP_349675 Clostridiumacetobutylicum buk2 20137415 Q97II1 Clostridium acetobutylicum

No enzyme required—Spontaneous cyclization. 6-Aminocaproyl-CoA willcyclize spontaneously to caprolactam, thus eliminating the need for adedicated enzyme for this step. A similar spontaneous cyclization isobserved with 4-aminobutyryl-CoA which forms pyrrolidinone (Ohsugi etal., J Biol Chem 256:7642-7651 (1981)).

Example XIII Preparation of a 6-Aminocaproic Acid Producing MicrobialOrganism Having a Pathway for Converting Acetyl-CoA and4-Aminobutyryl-CoA to 6-Aminocaproic Acid

This example describes the generation of a microbial organism capable ofproducing 6-aminocaproic acid from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the6-aminocaproic acid pathway shown in FIG. 11 that starts from acetyl-CoAand 4-aminobutyryl-CoA. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing6-aminocaproic acid. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproic acid,nucleic acids encoding the requisite enzymes are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaI(NP_415915.1), paaH (NP_415913.1), and maoC (NP_415905.1) genes encodingthe 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoAreductase, 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities,respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. In addition, the bcd(NP_349317.1), etfAB (NP_349315.1 and NP_349316.1), and acot8 (CAA15502)genes encoding 6-aminohex-2-enoyl-CoA reductase and 6-aminocaproyl-CoAhydrolase activities are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD(NP_904963.1), gabT (NP_417148.1), and cat2 (P38942.2) genes encodingsuccinyl-CoA reductase (aldehyde forming), GABA transaminase, and4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into athird compatible plasmid, pZS23, under the PA1/lacO promoter, toincrease the availability of 4-aminobutyryl-CoA. pZS23 is obtained byreplacing the ampicillin resistance module of the pZS13 vector(Expressys, Ruelzheim, Germany) with a kanamycin resistance module bywell-known molecular biology techniques. The three sets of plasmids aretransformed into E. coli strain MG1655 to express the proteins andenzymes required for 6-aminocaproic acid synthesis.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of the6-aminocaproic acid synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce6-aminocaproic acid is confirmed using HPLC, gas chromatography-massspectrometry (GCMS) and/or liquid chromatography-mass spectrometry(LCMS).

Microbial strains engineered to have a functional 6-aminocaproic acidsynthesis pathway are further augmented by optimization for efficientutilization of the pathway. Briefly, the engineered strain is assessedto determine whether any of the exogenous genes are expressed at a ratelimiting level. Expression is increased for any enzymes expressed at lowlevels that can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 6-aminocaproic acid. One modelingmethod is the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production of6-aminocaproic acid. Adaptive evolution also can be used to generatebetter producers of, for example, the acetyl-CoA and succinyl-CoAintermediates of the 6-aminocaproic acid product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe 6-aminocaproic acid producer to further increase production.

For large-scale production of 6-aminocaproic acid, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XIV Preparation of a 6-Aminocaproic Acid Producing MicrobialOrganism Having a Pathway for Converting Acetyl-CoA and4-Aminobutyryl-CoA to 6-Aminocaproic Acid

This example describes the generation of a microbial organism capable ofproducing 6-aminocaproic acid from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer the6-aminocaproic acid pathway shown in FIG. 11 that starts from acetyl-CoAand 4-aminobutyryl-CoA. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing6-aminocaproic acid. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproic acid,nucleic acids encoding the requisite enzymes are expressed in E. coliusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ(NP_415915.1), pcaIJ (AAN69545.1 and NP_746082.1), and bdh (AAA58352.1)genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase,3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoatereductase activities, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Inaddition, the enr (CAA76083.1) and hmd (ABC88407.1) genes encoding6-aminohex-2-enoate reductase and 3-hydroxy-6-aminohexanoate dehydrataseactivities are cloned into the pZA33 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. Lastly, the sucD (NP_904963.1),gabT (NP_417148.1), and cat2 (P38942.2) genes encoding succinyl-CoAreductase (aldehyde forming), GABA transaminase, and4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into athird compatible plasmid, pZS23, under the PA1/lacO promoter, toincrease the availability of 4-aminobutyryl-CoA. pZS23 is obtained byreplacing the ampicillin resistance module of the pZS13 vector(Expressys, Ruelzheim, Germany) with a kanamycin resistance module bywell-known molecular biology techniques. The three sets of plasmids aretransformed into E. coli strain MG1655 to express the proteins andenzymes required for 6-aminocaproic acid synthesis.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of the6-aminocaproic acid synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produce6-aminocaproic acid is confirmed using HPLC, gas chromatography-massspectrometry (GCMS) and/or liquid chromatography-mass spectrometry(LCMS).

Microbial strains engineered to have a functional 6-aminocaproic acidsynthesis pathway are further augmented by optimization for efficientutilization of the pathway. Briefly, the engineered strain is assessedto determine whether any of the exogenous genes are expressed at a ratelimiting level. Expression is increased for any enzymes expressed at lowlevels that can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 6-aminocaproic acid. One modelingmethod is the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production of6-aminocaproic acid. Adaptive evolution also can be used to generatebetter producers of, for example, the acetyl-CoA and succinyl-CoAintermediates of the 6-aminocaproic acid product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe 6-aminocaproic acid producer to further increase production.

For large-scale production of 6-aminocaproic acid, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XV Preparation of a Caprolactam Producing Microbial OrganismHaving a Pathway for Converting Acetyl-CoA and Succinyl-CoA to6-Aminocaproic acid

This example describes the generation of a microbial organism capable ofproducing caprolactam from acetyl-CoA and succinyl-CoA.

Escherichia coli is used as a target organism to engineer thecaprolactam pathway shown in FIG. 10 that starts from acetyl-CoA andsuccinyl-CoA. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing caprolactam.E. coli is amenable to genetic manipulation and is known to be capableof producing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce caprolactam, nucleicacids encoding the requisite enzymes are expressed in E. coli using wellknown molecular biology techniques (see, for example, Sambrook, supra,2001; Ausubel, supra, 1999). In particular, the paaI (NP_415915.1), paaH(NP_415913.1), and maoC (NP_415905.1) genes encoding the 3-oxoadipyl-CoAthiolase, 3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydrataseactivities, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd(NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.Lastly, the acr1 (YP_047869.1), gabT (NP_417148.1), and bioW(NP_390902.2) genes encoding adipyl-CoA reductase (aldehyde forming),6-aminocaproic acid transaminase, and 6-aminocaproyl-CoA synthaseactivities are cloned into a third compatible plasmid, pZS23, under thePA1/lacO promoter. pZS23 is obtained by replacing the ampicillinresistance module of the pZS13 vector (Expressys, Ruelzheim, Germany)with a kanamycin resistance module by well-known molecular biologytechniques. The three sets of plasmids are transformed into E. colistrain MG1655 to express the proteins and enzymes required forcaprolactam synthesis.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of thecaprolactam synthesis genes is corroborated using methods well known inthe art for determining polypeptide expression or enzymatic activity,including for example, Northern blots, PCR amplification of mRNA,immunoblotting, and the like. Enzymatic activities of the expressedenzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to producecaprolactam is confirmed using HPLC, gas chromatography-massspectrometry (GCMS) and/or liquid chromatography-mass spectrometry(LCMS).

Microbial strains engineered to have a functional caprolactam synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of caprolactam. One modeling method isthe bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of caprolactam.Adaptive evolution also can be used to generate better producers of, forexample, the acetyl-CoA and succinyl-CoA intermediates of thecaprolactam product. Adaptive evolution is performed to improve bothgrowth and production characteristics (Fong and Palsson, Nat. Genet.36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Basedon the results, subsequent rounds of modeling, genetic engineering andadaptive evolution can be applied to the caprolactam producer to furtherincrease production.

For large-scale production of caprolactam, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂₅₀₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XVI Preparation of a Hexamethylenediamine Producing MicrobialOrganism Having a Pathway for Converting Acetyl-CoA and Succinyl-CoA to6-Aminocaproic acid

This example describes the generation of a microbial organism capable ofproducing hexamethylenediamine from acetyl-CoA and succinyl-CoA.

Escherichia coli is used as a target organism to engineer thehexamethylenediamine pathway shown in FIG. 10 that starts fromacetyl-CoA and succinyl-CoA. E. coli provides a good host for generatinga non-naturally occurring microorganism capable of producinghexamethylenediamine. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to producehexamethylenediamine, nucleic acids encoding the requisite enzymes areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel, supra, 1999). Inparticular, the paaI (NP_415915.1), paaH (NP_415913.1), and maoC(NP_415905.1) genes encoding the 3-oxoadipyl-CoA thiolase,3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydrataseactivities, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd(NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.Lastly, the acr1 (YP_047869.1), gabT (NP_417148.1), bioW (NP_390902.2),and ygjG (NP_417544) genes encoding adipyl-CoA reductase (aldehydeforming), 6-aminocaproyl-CoA reductase (aldehyde forming),6-aminocaproic acid transaminase, 6-aminocaproyl-CoA synthase, andhexamethylenediamine transaminase activities are cloned into a thirdcompatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 isobtained by replacing the ampicillin resistance module of the pZS13vector (Expressys, Ruelzheim, Germany) with a kanamycin resistancemodule by well-known molecular biology techniques. The three sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for hexamethylenediamine synthesis.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of thehexamethylenediamine synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to producehexamethylenediamine is confirmed using HPLC, gas chromatography-massspectrometry (GCMS) and/or liquid chromatography-mass spectrometry(LCMS).

Microbial strains engineered to have a functional hexamethylenediaminesynthesis pathway are further augmented by optimization for efficientutilization of the pathway. Briefly, the engineered strain is assessedto determine whether any of the exogenous genes are expressed at a ratelimiting level. Expression is increased for any enzymes expressed at lowlevels that can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of hexamethylenediamine. One modelingmethod is the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production ofhexamethylenediamine. Adaptive evolution also can be used to generatebetter producers of, for example, the acetyl-CoA and succinyl-CoAintermediates of the hexamethylenediamine product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe hexamethylenediamine producer to further increase production.

For large-scale production of hexamethylenediamine, the above organismis cultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XVII Preparation of a Caprolactam Producing Microbial OrganismHaving a Pathway for Converting Acetyl-CoA and 4-aminobutyryl-CoA to6-Aminocaproyl-CoA

This example describes the generation of a microbial organism capable ofproducing caprolactam from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer thecaprolactam pathway shown in FIG. 11 that starts from acetyl-CoA and4-aminobutyryl-CoA. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing caprolactam.E. coli is amenable to genetic manipulation and is known to be capableof producing various products, like ethanol, acetic acid, formic acid,lactic acid, and succinic acid, effectively under anaerobic ormicroaerobic conditions.

To generate an E. coli strain engineered to produce caprolactam, nucleicacids encoding the requisite enzymes are expressed in E. coli using wellknown molecular biology techniques (see, for example, Sambrook, supra,2001; Ausubel, supra, 1999). In particular, the paaI (NP_415915.1), paaH(NP_415913.1), and maoC (NP_415905.1) genes encoding the3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase,3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, arecloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under thePA1/lacO promoter. In addition, the bcd (NP_349317.1) and etfAB(NP_349315.1 and NP_349316.1) genes encoding 6-aminohex-2-enoyl-CoAreductase activity are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD(NP_904963.1), gabT (NP_417148.1), and cat2 (P38942.2) genes encodingsuccinyl-CoA reductase (aldehyde forming), GABA transaminase, and4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into athird compatible plasmid, pZS23, under the PA1/lacO promoter, toincrease the availability of 4-aminobutyryl-CoA. pZS23 is obtained byreplacing the ampicillin resistance module of the pZS13 vector(Expressys, Ruelzheim, Germany) with a kanamycin resistance module bywell-known molecular biology techniques. The three sets of plasmids aretransformed into E. coli strain MG1655 to express the proteins andenzymes required for caprolactam synthesis.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of thecaprolactam synthesis genes is corroborated using methods well known inthe art for determining polypeptide expression or enzymatic activity,including for example, Northern blots, PCR amplification of mRNA,immunoblotting, and the like. Enzymatic activities of the expressedenzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to producecaprolactam is confirmed using HPLC, gas chromatography-massspectrometry (GCMS) and/or liquid chromatography-mass spectrometry(LCMS).

Microbial strains engineered to have a functional caprolactam synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of caprolactam. One modeling method isthe bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of caprolactam.Adaptive evolution also can be used to generate better producers of, forexample, the acetyl-CoA and succinyl-CoA intermediates of thecaprolactam product. Adaptive evolution is performed to improve bothgrowth and production characteristics (Fong and Palsson, Nat. Genet.36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Basedon the results, subsequent rounds of modeling, genetic engineering andadaptive evolution can be applied to the caprolactam producer to furtherincrease production.

For large-scale production of caprolactam, the above organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XVIII Preparation of a Hexamethylenediamine Producing MicrobialOrganism Having a Pathway for Converting Acetyl-CoA and4-aminobutyryl-CoA to 6-Aminocaproyl-CoA

This example describes the generation of a microbial organism capable ofproducing hexamethylenediamine from acetyl-CoA and 4-aminobutyryl-CoA.

Escherichia coli is used as a target organism to engineer thehexamethylenediamine pathway shown in Figure XVII that starts fromacetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host forgenerating a non-naturally occurring microorganism capable of producinghexamethylenediamine. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to producehexamethylenediamine, nucleic acids encoding the requisite enzymes areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel, supra, 1999). Inparticular, the paaI (NP_415915.1), paaH (NP_415913.1), and maoC(NP_415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase,3-oxo-6-aminohexanoyl-CoA reductase, 3-hydroxy-6-aminohexanoyl-CoAdehydratase activities, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Inaddition, the bcd (NP_349317.1), etfAB (NP_349315.1 and NP_349316.1),acr1 (YP_047869.1), and ygjG (NP_417544) genes encoding6-aminohex-2-enoyl-CoA reductase, 6-aminocaproyl-CoA reductase (aldehydeforming), and hexamethylenediamine transaminase activities are clonedinto the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. Lastly, the sucD (NP_904963.1), gabT (NP_417148.1), and cat2(P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming),GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferaseactivities are cloned into a third compatible plasmid, pZS23, under thePA1/lacO promoter, to increase the availability of 4-aminobutyryl-CoA.pZS23 is obtained by replacing the ampicillin resistance module of thepZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistancemodule by well-known molecular biology techniques. The three sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for hexamethylenediamine synthesis.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of thehexamethylenediamine synthesis genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to producehexamethylenediamine is confirmed using HPLC, gas chromatography-massspectrometry (GCMS) and/or liquid chromatography-mass spectrometry(LCMS).

Microbial strains engineered to have a functional hexamethylenediaminesynthesis pathway are further augmented by optimization for efficientutilization of the pathway. Briefly, the engineered strain is assessedto determine whether any of the exogenous genes are expressed at a ratelimiting level. Expression is increased for any enzymes expressed at lowlevels that can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of hexamethylenediamine. One modelingmethod is the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production ofhexamethylenediamine. Adaptive evolution also can be used to generatebetter producers of, for example, the acetyl-CoA and succinyl-CoAintermediates of the hexamethylenediamine product. Adaptive evolution isperformed to improve both growth and production characteristics (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science314:1565-1568 (2006)). Based on the results, subsequent rounds ofmodeling, genetic engineering and adaptive evolution can be applied tothe hexamethylenediamine producer to further increase production.

For large-scale production of hexamethylenediamine, the above organismis cultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing the culture vessel, for example, flasks can be sealed witha septum and crimp-cap. Microaerobic conditions also can be utilized byproviding a small hole in the septum for limited aeration. The pH of themedium is maintained at a pH of around 7 by addition of an acid, such asH₂SO₄. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm) and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu, Columbia Md.), for example, using an Aminex® series of HPLCcolumns (for example, HPX-87 series) (BioRad, Hercules Calif.), using arefractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XIX Pathways for Production of 6-Aminocaproic Acid from SuccinicSemialdehyde and Pyruvate

This example describes exemplary pathways for production of6-aminocaproic acid. Novel pathways for producing 6-aminocaproic acid(6-ACA) and related products are described herein. These pathwayssynthesize 6-ACA from succinic semialdehyde and pyruvate, utilizingaldolase and hydratase enzymes from the 4-hydroxyphenylacetic aciddegradation pathway. The candidate enzymes, and associated risks ofimplementation are discussed in Example XXI below.

This invention is directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyze 6-ACAproduction. Successfully engineering these pathways entails identifyingan appropriate set of enzymes with sufficient activity and specificity,cloning their corresponding genes into a production host, optimizing theexpression of these genes in the production host, optimizingfermentation conditions, and assaying for product formation followingfermentation.

6-aminocaproic acid and derivatives are produced from succinicsemialdehyde and pyruvate in a minimum of five enzymatic steps. In thefirst step of all pathways, pyruvate and succinic semialdehyde arejoined by 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase. The productof this reaction, HODH, is then dehydrated by 2-oxohept-4-ene-1,7-dioate(OHED) hydratase to form OHED In subsequent steps, OHED istransaminated, decarboxylated or reduced as shown in FIG. 12.

In one route, the alkene of OHED is reduced by OHED reductase, forming2-oxoheptane-1,7-dioate (2-OHD) (FIG. 12, Step C), a 2-ketoacid. 2-OHDis then converted to adipate semialdehyde by a ketoacid decarboxylase(FIG. 12, Step D). In the final step, the aldehyde of adipatesemialdehyde is converted to an amine by an aminotransferase or anaminating oxidoreductase (FIG. 12, Step E).

In a similar route, the 2-keto group of 2-OHD is transaminated by anaminotransferase or an aminating oxidoreductase (FIG. 12, Step H) toform 2-aminoheptane-1,7-dioate (2-AHD). This product is thendecarboxylated by 2-AHD decarboxylase to form 6-aminocapropate (FIG. 12,Step I).

In an alternate route, OHED is first decarboxylated by OHEDdecarboxylase (FIG. 12, Step F), resulting in the formation of6-oxohex-4-enoate (6-OHE). The alkenal group of 6-OHE is reduced by anoxidoreductase to adipate semialdehyde (FIG. 12, Step G). Adipatesemialdehyde is then converted to 6-aminocaproate by an aminotransferaseor aminating oxidoreductase (FIG. 12, Step E).

Yet another route calls for an aminotransferase or aminatingoxidoreductase to convert OHED to 2-aminohept-4-ene-1,7-dioate (2-AHE)(FIG. 12, Step J). The alkene of 2-AHE is subsequently reduced by analkene oxidoreductase (FIG. 12, Step K). The product of this reaction,2-AHD, is then decarboxylated by an amino acid decarboxylase (FIG. 12,Step I) to form 6-aminocaproate.

In yet another route, HODH is converted to 3-hydroxyadipyl-CoA by eitheran HODH dehydrogenase or and HODH formate-lyase (FIG. 12, Step L).3-Hydroxyadipyl-CoA is subsequently dehydrated and reduced to formadipyl-CoA (FIG. 12, Steps M, N). Adipyl-CoA is reduced and de-acylatedto form adipate semialdehyde (FIG. 12, Step O), which is then convertedto 6-aminocaproate by an aminotransferase or an aminating oxidoreductase(FIG. 12, Step E).

In a similar route, HODH is first converted to OHED (FIG. 12, Step B),as described above. OHED is then converted to 2,3-dehydroadipyl-CoA by adehydrogenase or an OHED formate-lyase (FIG. 12, Step P).2,3-Dihydroadipyl-CoA is then reduced to adipyl-CoA (FIG. 12, Step N),which is converted to 6-aminocaproate via adipate semialdehyde (FIG. 12,Steps 0, E), as described previously.

In the final route, HODH is converted to 2-OHD via steps B and C, asdescribed previously. A 2-OHD formate-lyase or dehydrogenase converts2-OHD to adipyl-CoA (FIG. 12, Step Q), which is then reduced by aCoA-dependent aldehyde dehydrogenase (FIG. 12, Step O). The product,adipate semialdehyde, is converted to 6-aminocaproate by anaminotransferase or aminating oxidoreductase (FIG. 12, Step E).

The routes detailed in FIG. 12 are able to achieve the maximumtheoretical 6-ACA yield of 0.8 moles 6-ACA per mole glucose utilized.The energetic yield is also favorable, with a maximum of 1.6 moles ATPper mole glucose utilized at the maximum product yield. The followingassumptions were used to calculate yield: 1) phosphoenolpyruvate (PEP)carboxykinase is able to operate in the ATP-generating direction, 2) NH4and 6-ACA are transported into the cell by proton antiport, and 3)succinic semialdehyde is formed from alpha-ketoglutarate and/orsuccinyl-CoA. Succinic semialdehyde dehydrogenase is a NAD(P)H andCoA-dependent aldehyde dehydrogenase that converts succinyl-CoA tosuccinic semialdehyde. Succinic semialdehyde is formed fromalpha-ketoglutarate by two enzymes: alpha-ketoglutarate decarboxylaseand 4-aminobutyrate transaminase.

Example XX Pathways for Production of Hexamethylenediamine from6-Aminocaproate

This example describes exemplary pathways for production ofhexamethylenediamine.

Novel pathways for producing hexamethylenediamine (HMDA) and relatedproducts are described herein. This pathway synthesizes HMDA from6-Aminocaproate (6-ACA). These pathways involve activation of the acidgroup by phosphorylation and/or acylation. Acetylation of the terminalamino group provides protection from spontaneous cyclization of pathwayintermediates. The candidate enzymes, and associated risks ofimplementation are discussed in Example XXI below.

This invention is directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyze HMDAproduction. Successfully engineering these pathways entails identifyingan appropriate set of enzymes with sufficient activity and specificity,cloning their corresponding genes into a production host, optimizing theexpression of these genes in the production host, optimizingfermentation conditions, and assaying for product formation followingfermentation.

Several pathways for producing HMDA from 6-aminocaproate are detailed inFIG. 13. All routes entail activation of the carboxylic acid group,followed by reduction and transamination. In three routes,6-aminocaproate is activated directly while in other routes, theterminal amine group is protected by N-acetylation to preventspontaneous cyclization.

In one route, 6-aminocaproate is phosphorylated to 6-AHOP by6-aminocaproate kinase (FIG. 13, Step A). 6-AHOP is then reduced to6-aminocaproic semialdehyde (FIG. 13, Step B) and subsequentlytransaminated (FIG. 13, Step C) by an aminotransferase or an aminatingoxidoreductase.

Alternately, 6-AHOP is converted to 6-aminocaproyl-CoA by anacyltransferase (FIG. 13, Step L). 6-Aminocaproyl-CoA is then reduced to6-aminocaproic semialdehyde by a CoA-dependent aldehyde dehydrogenase(FIG. 13, Step N). HMDA is then formed by transamination of6-aminocaproic semialdehyde by an aminotransferase or aminatingoxidoreductase (FIG. 13, Step C).

In yet another route, 6-aminocaproate is first activated to a CoAderivative by a CoA transferase or CoA ligase (FIG. 13, Step M). Theproduct, 6-aminocaproyl-CoA, may spontaneously cyclize, or be convertedto 6-aminocaproic semialdehyde by an aldehyde-forming CoA-dependentaldehyde dehydrogenase (FIG. 13, Step N). 6-Aminocaproic semialdehyde isconverted to HMDA by an aminotransferase or an aminating oxidoreductase(FIG. 13, Step C).

Additional routes proceed from 6-acetamidohexanoate, the acetylatedproduct of 6-aminocaproate N-acetyltransferase. 6-Acetamidohexanoate isconverted to 6-acetamidohexanal by different routes (described below).In the final two steps of these routes, 6-acetamidohexanal is firstconverted to 6-acetamidohexanamine by an aminotransferase or anaminating oxidoreductase (FIG. 13, Step G). 6-Acetamidohexanamine issubsequently converted to HMDA by an amide hydrolase or anN-acetyltransferase (FIG. 13, Step H).

In one route, 6-acetamidohexanoate is phosphorylated by6-acetamidohexanoate kinase (FIG. 13, Step E). The product, 6-AAHOP, isreduced to form 6-acetamidohexanal (FIG. 13, Step F), which is thenconverted to HMDA as described above.

In another route, 6-acetamidohexanoate is activated to6-acetamidohexanoyl-CoA by a CoA transferase or CoA ligase (FIG. 13,Step I). The CoA derivative is then reduced to 6-acetamidohexanal by analdehyde-forming CoA-dependent oxidoreductase (FIG. 13, Step J).6-acetamidohexanal is then converted to HMDA as described above.

Alternately, 6-acetamidohexanoate is phosphorylated to 6-AAHOP (FIG. 13,Step E) and subsequently converted to 6-acetamidohexanoyl-CoA by anacyltransferase (FIG. 13, Step K). 6-Acetamidohexanoyl-CoA is thenreduced to HMDA as described previously.

Example XXI Enzyme Classification System for Production of6-Aminocaproic Acid and Hexamethylenediamine

This example describes the enzyme classification system for theexemplary pathways described in Examples XIX and XX for production of6-aminocaproate or hexamethylenediamine.

All transformations depicted in FIGS. 12 and 13 fall into the generalcategories of transformations shown in Table 9. Below is described anumber of biochemically characterized genes in each category.Specifically listed are genes that can be applied to catalyze theappropriate transformations in FIGS. 12-13 when properly cloned andexpressed.

Table 9 shows the enzyme types useful to convert common centralmetabolic intermediates into 6-aminocaproate and hexamethylenediamine.The first three digits of each label correspond to the first threeEnzyme Commission number digits which denote the general type oftransformation independent of substrate specificity.

TABLE 9 LABEL FUNCTION 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde)1.2.1.c Oxidoreductase (2-ketoacid to acyl-CoA) 1.2.1.d Oxidoreductase(phosphonic acid to aldehyde) 1.3.1.a Oxidoreductase (alkene to alkane)1.4.1.a Oxidoreductase (ketone or aldehyde to amino) 2.3.1.aAcyltransferase (transferring CoA to phospho) 2.3.1.c Acyltransferase(N-acetyltransferase) 2.3.1.d Acyltransferase (formateC-acyltransferase) 2.6.1.a Aminotransferase 2.7.2.a Phosphotransferase(carboxy acceptor) 2.8.3.a Coenzyme-A transferase 3.5.1.a Hydrolase(acting on linear amides) 4.1.1.a Carboxy-lyase 4.1.2.a Aldehyde-lyase4.2.1.a Hydro-lyase 6.2.1.a Acid-thiol ligase

1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde).

The transformations of 6-acetamidohexanoyl-CoA to 6-acetamidohexanal(FIG. 13, Step J) and 6-aminocaproyl-CoA to 6-aminocaproic semialdehyde(FIG. 13, Step N) are catalyzed by CoA-dependent oxidoreductase enzymein the EC class 1.2.1. Adipyl-CoA is converted to adipate semialdehydeby adipyl-CoA oxidoreductase, an enzyme with similar functionality (FIG.12, Step O). Succinic semialdehyde dehydrogenase, an enzyme that formsFIG. 12 precursor succinic semialdehyde from succinyl-CoA, is also aCoA-dependent oxidoreductase. Oxidoreductases in the EC class 1.2.1.-are capable of reducing an acyl-CoA to its corresponding aldehyde.Exemplary genes that encode such enzymes include the Acinetobactercalcoaceticus acr1 encoding a fatty acyl-CoA reductase (Reiser andSomerville, Journal of Bacteriology 179:2969-2975 (1997)), theAcinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependentsuccinic semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)). SucD of P. gingivalis is another succinic semialdehydedehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)).The acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded bybphG, is yet another candidate as it has been demonstrated to oxidizeand acylate acetaldehyde, propionaldehyde, butyraldehyde,isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol.175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, theenzyme encoded by adhE in Leuconostoc mesenteroides has been shown tooxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA(Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo etal., Biotechnol Lett. 27:505-510 (2005)).

Gene GenBank name GI# Accession # Organism acr1 50086359 YP_047869.1Acinetobacter calcoaceticus acr1 1684886 AAC45217 Acinetobacter baylyiacr1 18857901 BAB85476.1 Acinetobacter sp. Strain M-1 sucD 172046062P38947.1 Clostridium kluyveri sucD 34540484 NP_904963.1 Porphyromonasgingivalis bphG 425213 BAA03892.1 Pseudomonas sp adhE 55818563AAV66076.1 Leuconostoc mesenteroides

An additional enzyme that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786(2007); and Thauer, R. K., Science. 318:1732-1733 (2007)). The enzymeutilizes NADPH as a cofactor and has been characterized inMetallosphaera and Sulfolobus sp (Alber et al., J. Bacteriol.188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410(2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula(Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al.,Science. 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductasefrom Sulfolobus tokodaii was cloned and heterologously expressed in E.coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme hasalso been shown to catalyze the conversion of methylmalonyl-CoA to itscorresponding aldehyde (WIPO Patent Application WO/2007/141208 KindCode: A2). Although the aldehyde dehydrogenase functionality of theseenzymes is similar to the bifunctional dehydrogenase from Chloroflexusaurantiacus, there is little sequence similarity. Both malonyl-CoAreductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius and have been listed below.Yet another candidate for CoA-acylating aldehyde dehydrogenase is theald gene from Clostridium beijerinckii (Toth et al., Appl EnvironMicrobiol 65:4973-4980 (1999)). This enzyme has been reported to reduceacetyl-CoA and butyryl-CoA to their corresponding aldehydes. This geneis very similar to eutE that encodes acetaldehyde dehydrogenase ofSalmonella typhimurium and E. coli (Toth et al., Appl Environ Microbiol65:4973-4980 (1999)).

GenBank Gene name GI# Accession # Organism Msed_0709 146303492YP_001190808.1 Metallosphaera sedula mcr 15922498 NP_378167.1 Sulfolobustokodaii asd-2 15898958 NP_343563.1 Sulfolobus solfataricus Saci_237070608071 YP_256941.1 Sulfolobus acidocaldarius Ald 49473535 AAT66436Clostridium beijerinckii eutE 687645 AAA80209 Salmonella typhimuriumeutE 2498347 P77445 Escherichia coli

1.2.1.c Oxidoreductase (2-Ketoacid to Acyl-CoA).

Several transformations in FIG. 12 require conversion of a 2-ketoacid toan acyl-CoA (Steps L, P and Q) by an enzyme in the EC class 1.2.1. Suchreactions are catalyzed by multi-enzyme complexes that catalyze a seriesof partial reactions which result in acylating oxidative decarboxylationof 2-keto-acids. Exemplary enzymes include 1) branched-chain 2-keto-aciddehydrogenase, 2) alpha-ketoglutarate dehydrogenase, and 3) the pyruvatedehydrogenase multienzyme complex (PDHC). Each of the 2-keto-aciddehydrogenase complexes occupies key positions in intermediarymetabolism, and enzyme activity is typically tightly regulated (Fries etal., Biochemistry 42:6996-7002 (2003)). The enzymes share a complex butcommon structure composed of multiple copies of three catalyticcomponents: alpha-ketoacid decarboxylase (E1), dihydrolipoamideacyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). The E3component is shared among all 2-keto-acid dehydrogenase complexes in anorganism, while the E1 and E2 components are encoded by different genes.The enzyme components are present in numerous copies in the complex andutilize multiple cofactors to catalyze a directed sequence of reactionsvia substrate channeling. The overall size of these dehydrogenasecomplexes is very large, with molecular masses between 4 and 10 millionDa (i.e. larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normallylow or limited under anaerobic conditions in E. coli. Increasedproduction of NADH (or NADPH) could lead to a redox-imbalance, and NADHitself serves as an inhibitor to enzyme function. Engineering effortshave increased the anaerobic activity of the E. coli pyruvatedehydrogenase complex (Kim et al., Appl. Environ. Microbiol.73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008); andZhou et al., Biotechnol. Lett. 30:335-342 (2008)). For example, theinhibitory effect of NADH can be overcome by engineering an H₃₂₂Ymutation in the E3 component (Kim et al., J. Bacteriol. 190:3851-3858(2008)). Structural studies of individual components and how they worktogether in complex provide insight into the catalytic mechanisms andarchitecture of enzymes in this family (Aevarsson et al., Nat. Struct.Biol. 6:785-792 (1999); and Zhou et al., Proc. Natl. Acad. Sci. U.S.A98:14802-14807 (2001)). The substrate specificity of the dehydrogenasecomplexes varies in different organisms, but generally branched-chainketo-acid dehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278(1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD geneexpression is downregulated under anaerobic conditions and during growthon glucose (Park et al., Mol. Microbiol. 15:473-482 (1995)). Althoughthe substrate range of AKGD is narrow, structural studies of thecatalytic core of the E2 component pinpoint specific residuesresponsible for substrate specificity (Knapp et al., J. Mol. Biol.280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB (E1and E2) and pdhD (E3, shared domain), is regulated at thetranscriptional level and is dependent on the carbon source and growthphase of the organism (Resnekov et al., Mol. Gen. Genet. 234:285-296(1992)). In yeast, the LPD1 gene encoding the E3 component is regulatedat the transcriptional level by glucose (Roy and Dawes, J. Gen.Microbiol. 133:925-933 (1987)). The E1 component, encoded by KGD1, isalso regulated by glucose and activated by the products of HAP2 and HAP3(Repetto and Tzagoloff, Mol. Cell Biol. 9:2695-2705 (1989)). The AKGDenzyme complex, inhibited by products NADH and succinyl-CoA, iswell-studied in mammalian systems, as impaired function of has beenlinked to several neurological diseases (Tretter and dam-Vizi, Philos.Trans. R. Soc. Lond B Biol. Sci. 360:2335-2345 (2005)).

GenBank Gene name GI# Accession # Organism sucA 16128701 NP_415254.1Escherichia coli sucB 16128702 NP_415255.1 Escherichia coli lpd 16128109NP_414658.1 Escherichia coli odhA 51704265 P23129.2 Bacillus subtilisodhB 129041 P16263.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillussubtilis KGD1 6322066 NP_012141.1 Saccharomyces cerevisiae KGD2 6320352NP_010432.1 Saccharomyces cerevisiae LPD1 14318501 NP_116635.1Saccharomyces cerevisiae

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as2-oxoisovalerate dehydrogenase, participates in branched-chain aminoacid degradation pathways, converting 2-keto acids derivatives ofvaline, leucine and isoleucine to their acyl-CoA derivatives and CO₂.The complex has been studied in many organisms including Bacillussubtilis (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)), Rattusnorvegicus (Namba et al., J. Biol. Chem. 244:4437-4447 (1969)) andPseudomonas putida (Sokatch et al., J. Bacteriol. 148:647-652 (1981)).In Bacillus subtilis the enzyme is encoded by genes pdhD (E3 component),bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al.,Eur. J. Biochem. 213:1091-1099 (1993)). In mammals, the complex isregulated by phosphorylation by specific phosphatases and proteinkinases. The complex has been studied in rat hepatocites (Chicco et al.,J. Biol. Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha(E1 alpha), Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3components of the Pseudomonas putida BCKAD complex have beencrystallized (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); andMattevi et al., Science. 255:1544-1550 (1992)) and the enzyme complexhas been studied (Sokatch et al., J. Bacteriol. 148:647-652 (1981)).Transcription of the P. putida BCKAD genes is activated by the geneproduct of bkdR (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)).In some organisms including Rattus norvegicus (Paxton et al., Biochem.J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al.,Biochem. Mol. Biol. Int. 31:911-922 (1993)), this complex has been shownto have a broad substrate range that includes linear oxo-acids such as2-oxobutanoate and alpha-ketoglutarate, in addition to thebranched-chain amino acid precursors. The active site of the bovineBCKAD was engineered to favor alternate substrate acetyl-CoA (Meng andChuang, Biochemistry. 33:12879-12885 (1994)).

GenBank Gene name GI# Accession # Organism bfmBB 16079459 NP_390283.1Bacillus subtilis bfmBAA 16079461 NP_390285.1 Bacillus subtilis bfmBAB16079460 NP_390284.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillussubtilis lpdV 118677 P09063.1 Pseudomonas putida bkdB 129044 P09062.1Pseudomonas putida bkdA1 26991090 NP_746515.1 Pseudomonas putida bkdA226991091 NP_746516.1 Pseudomonas putida Bckdha 77736548 NP_036914.1Rattus norvegicus Bckdhb 158749538 NP_062140.1 Rattus norvegicus Dbt158749632 NP_445764.1 Rattus norvegicus Dld 40786469 NP_955417.1 Rattusnorvegicus

The pyruvate dehydrogenase complex, catalyzing the conversion ofpyruvate to acetyl-CoA, has also been extensively studied. In the E.coli enzyme, specific residues in the E1 component are responsible forsubstrate specificity (Bisswanger, J Biol Chem. 256:815-822 (1981);Bremer, Eur. J Biochem. 8:535-540 (1969); and Gong et al., J Biol Chem.275:13645-13653 (2000)). As mentioned previously, enzyme engineeringefforts have improved the E. coli PDH enzyme activity under anaerobicconditions (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007);Kim et al., J Bacteriol. 190:3851-3858 (2008)); and Zhou et al.,Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH,the B. subtilis complex is active and required for growth underanaerobic conditions (Nakano et al., J Bacteriol. 179:6749-6755 (1997)).The Klebsiella pneumoniae PDH, characterized during growth on glycerol,is also active under anaerobic conditions (Menzel et al., J. Biotechnol.56:135-142 (1997)). Crystal structures of the enzyme complex from bovinekidney (Zhou et al., Proc. Natl. Acad. Sci. U.S.A 98:14802-14807 (2001))and the E2 catalytic domain from Azotobacter vinelandii are available(Mattevi et al., Science. 255:1544-1550 (1992)). Some mammalian PDHenzymes complexes can react on alternate substrates such as2-oxobutanoate, although comparative kinetics of Rattus norvegicus PDHand BCKAD indicate that BCKAD has higher activity on 2-oxobutanoate as asubstrate (Paxton et al., Biochem. J. 234:295-303 (1986)).

GenBank Gene name GI# Accession # Organism aceE 16128107 NP_414656.1Escherichia coli aceF 16128108 NP_414657.1 Escherichia coli lpd 16128109NP_414658.1 Escherichia coli pdhA 3123238 P21881.1 Bacillus subtilispdhB 129068 P21882.1 Bacillus subtilis pdhC 129054 P21883.2 Bacillussubtilis pdhD 118672 P21880.1 Bacillus subtilis aceE 152968699YP_001333808.1 Klebsiella pneumonia aceF 152968700 YP_001333809.1Klebsiella pneumonia lpdA 152968701 YP_001333810.1 Klebsiella pneumoniaPdha1 124430510 NP_001004072.2 Rattus norvegicus Pdha2 16758900NP_446446.1 Rattus norvegicus Dlat 78365255 NP_112287.1 Rattusnorvegicus Dld 40786469 NP_955417.1 Rattus norvegicus

As an alternative to the large multienzyme 2-keto-acid dehydrogenasecomplexes described above, some anaerobic organisms utilize enzymes inthe 2-ketoacid oxidoreductase family (OFOR) to catalyze acylatingoxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenasecomplexes, these enzymes contain iron-sulfur clusters, utilize differentcofactors, and use ferredoxin or flavodoxin as electron acceptors inlieu of NAD(P)H. While most enzymes in this family are specific topyruvate as a substrate (POR) some 2-keto-acid:ferredoxinoxidoreductases have been shown to accept a broad range of 2-ketoacidsas substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukudaand Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al.,J. Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from thethermoacidophilic archaeon Sulfolobus tokodaii 7, which contains analpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi,Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem.120:587-599 (1996)). A plasmid-based expression system has beendeveloped for efficiently expressing this protein in E. coli (Fukuda etal., Eur. Biochem. 268:5639-5646 (2001)) and residues involved insubstrate specificity were determined (Fukuda and Wakagi, Biochim.Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str.K1 have also been recently cloned into E. coli, characterized, and foundto react with a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett.579:2319-2322 (2005)). The gene sequences of these OFOR candidates areavailable, although they do not have GenBank identifiers assigned todate. There is bioinformatic evidence that similar enzymes are presentin all archaea, some anaerobic bacteria and amitochondrial eukarya(Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). Thisclass of enzyme is also interesting from an energetic standpoint, asreduced ferredoxin could be used to generate NADH by ferredoxin-NADreductase (Petitdemange et al., Biochim. Biophys. Acta 421:334-337(1976)). Also, since most of the enzymes are designed to operate underanaerobic conditions, less enzyme engineering may be required relativeto enzymes in the 2-keto-acid dehydrogenase complex family for activityin an anaerobic environment.

GenBank Gene name GI# Accession # Organism ST2300 15922633 NP_378302.1Sulfolobus tokodaii 7

1.2.1.d Oxidoreductase (Phosphonic Acid to Aldehyde).

The reduction of a phosphonic acid to its corresponding aldehyde iscatalyzed by an oxidoreductase in the EC class 1.2.1. Steps B and F inFIG. 13 require such an enzyme for the reduction of 6-AHOP and 6-AAHOPto their corresponding aldehydes. These reactions are not catalyzed byknown enzymes, but a similar reaction is catalyzed by aspartatesemialdehyde dehydrogenase (ASD, EC 1.2.1.11): the NADPH-dependentreduction of 4-aspartyl phosphate to aspartate-4-semialdehyde. ASDparticipates in amino acid biosynthesis and recently has been studied asan antimicrobial target (Hadfield et al., Biochemistry 40:14475-14483(2001)). The E. coli ASD structure has been solved (Hadfield et al., JMol. Biol. 289:991-1002 (1999)) and the enzyme has been shown to acceptthe alternate substrate beta-3-methylaspartyl phosphate (Shames et al.,J Biol. Chem. 259:15331-15339 (1984)). The Haemophilus influenzae enzymehas been the subject of enzyme engineering studies to alter substratebinding affinities at the active site (Blanco et al., Acta Crystallogr.D. Biol. Crystallogr. 60:1388-1395 (2004); and Blanco et al., ActaCrystallogr. D. Biol. Crystallogr. 60:1808-1815 (2004)). Other ASDcandidates are found in Mycobacterium tuberculosis (Shafiani et al., JAppl Microbiol 98:832-838 (2005)), Methanococcus jannaschii (Faehnle etal., J Mol. Biol. 353:1055-1068 (2005)), and the infectiousmicroorganisms Vibrio cholera and Heliobacter pylori (Moore et al.,Protein Expr. Purif. 25:189-194 (2002)). A related enzyme candidate isacetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme thatnaturally reduces acetylglutamylphosphate toacetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et al.,Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly and Devine,Microbiology 140 (Pt 5):1023-1025 (1994)) and other organisms.

GenBank Gene name GI# Accession # Organism Asd 16131307 NP_417891.1Escherichia coli Asd 68249223 YP_248335.1 Haemophilus influenzae Asd1899206 AAB49996 Mycobacterium tuberculosis VC2036 15642038 NP_231670Vibrio cholera Asd 210135348 YP_002301787.1 Heliobacter pylori ARG5, 66320913 NP_010992.1 Saccharomyces cerevisiae argC 16078184 NP_389001.1Bacillus subtilis

1.3.1.a Oxidoreductase (Alkene to Alkane).

Several transformations fall into the category of oxidoreductases thatreduce an alkene to an alkane (EC 1.3.1.-). For example, Steps C, G, Kand N in FIG. 12, catalyzed by OHED reductase, 6-OHE reductase, 2-AHEreductase and 2,3-dehydroadipyl-CoA reductase, respectively, fall intothis category. Enone reductase, alkenal reductase, and enoate reductaseenzymes are suitable enzyme candidates for catalyzing thetransformations of Steps C, G and K. Enoyl-CoA reductase enzymescatalyze the conversion of 2,3-dehydroadipyl-CoA to adipyl-CoA (Step N).

Enzymes with enone reductase activity have been identified inprokaryotes, eukaryotes and plants (Shimoda et al., Bulletin of thechemical Society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur.J Biochem. 255:271-278 (1998)). Two enone reductases from the cytosolicfraction of Saccharomyces cerevisiae were purified and characterized,and found to accept a variety of alkenals (similar to 6-OHE) and enoylketones (similar to OHED) as substrates (Wanner and Tressl, Eur. JBiochem. 255:271-278 (1998)). Genes encoding these enzymes have not beenidentified to date. Cell extracts of cyanobacterium Synechococcus sp.PCC7942 reduced a variety enone substrates to their corresponding alkylketones (Shimoda et al., Bulletin of the chemical Society of Japan77:2269-2 (2004)). Genes have not been associated with this activity inthis organism. Enone reductases in other organisms can also catalyzethis transformation.

A recombinant NADPH-dependent enone reductase from Nicotiana tabacum,encoded by NtRed1, was functionally expressed and characterized in E.coli (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). Thisreductase was functional on the exocyclic enoyl ketone pulegone(Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). An enzymecandidate in S. cerevisiae at the locus YML131W, bears 30% identity toNtRed1(evalue=1e-26). The amino acid sequence of NtRed1 sharessignificant homology with 2-alkenal reductase from Arabidopsis thaliana,zeta-crystallin homolog from A. thaliana, pulegone reductase from Menthepiperita and phenylpropenal alkene reductase from Pinus taeda. Theseenzymes are known to catalyze the reduction of alkenes ofα,β-unsaturated ketones and aldehydes.

GenBank Gene name GI# Accession # Organism NtRed1 6692816 BAA89423Nicotiana tabacum YML131W 45269874 AAS56318.1 Saccharomyces cerevisiaeAtDBR1 15237888 NP-197199 Arabidopsis thaliana P2 886430 CAA89262Arabidopsis thaliana PulR 34559418 AAQ75423 Menthe piperita PtPPDBR110816011 ABG91753 Pinus taeda

2-Alkenal reductase catalyzes the reduction of α, β-unsaturated doublebonds of aldehydes and ketones. A barley alkenal hydrogenase ALH1 wasidentified with activity for a range of α,β-unsaturated ketones andaldehydes including trans-2-nonenal, 2-hexenal, traumatin and1-octene-3-one (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721(2005)). The Hordeum vulgare ALH1 cDNA was cloned expressed in E. coli(Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)).

GenBank Gene name GI# Accession # Organism ALH1 62765876 AAX99161Hordeum vulgare ALH1 195652571 ACG45753 Zea mays

2-Enoate reductase enzymes are known to catalyze the NAD(P)H-dependentreduction of a wide variety of α, β-unsaturated carboxylic acids andaldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). In therecently published genome sequence of C. kluyveri, 9 coding sequencesfor enoate reductases were reported, out of which one has beencharacterized (Seedorf et al., Proc. Natl. Acad. Sci U.S.A 105:2128-2133(2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticumhave been cloned and sequenced and show 59% identity to each other. Theformer gene is also found to have approximately 75% similarity to thecharacterized gene in C. kluyveri (Giesel and Simon, Arch. Microbiol135:51-57 (1983)). It has been reported based on these sequence resultsthat enr is very similar to the dienoyl CoA reductase in E. coli (fadH)(Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). The C.thermoaceticum enr gene has also been expressed in a catalyticallyactive form in E. coli (Rohdich et al., J. Biol. Chem. 276:5779-5787(2001)).

GenBank Gene name GI# Accession # Organism enr 169405742 ACA54153.1Clostridium botulinum A3 str enr 2765041 CAA71086.1 Clostridiumtyrobutyricum enr 3402834 CAA76083.1 Clostridium kluyveri enr 83590886YP_430895.1 Moorella thermoacetica fadH 16130976 NP_417552.1 Escherichiacoli

Another candidate enoate reductase is 3-oxoadipate oxidoreductase(maleylacetate reductase), an enzyme catalyzing the reduction of2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. The enzymeactivity was identified and characterized in Pseudomonas sp. strain B13(Kaschabek and Reineke, J. Bacteriol. 177:320-325 (1995); and Kaschabek.and Reineke, J. Bacteriol. 175:6075-6081 (1993)), and the coding genewas cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803(1997)). Candidate genes for 3-oxoadipate oxidoreductase include cicEgene from Pseudomonas sp. strain B13 (Kasberg et al., J. Bacteriol.179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert etal., J. Bacteriol. 180:3503-3508 (1998)), and macA gene from Ralstoniaeutropha (also known as Cupriavidus necator) (Seibert et al.,Microbiology 150:463-472 (2004)).

GenBank Gene name GI# Accession # Organism clcE 3913241 O30847.1Pseudomonas sp. strain B13 macA 7387876 O84992.1 Rhodococcus opacus macA5916089 AAD55886 Cupriavidus necator

Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing thereduction of 2,3-dehydroadipyl-CoA to adipyl-CoA (FIG. 12, Step N). Oneexemplary enoyl-CoA reductase is the gene product of bcd from C.acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boyntonet al., J. Bacteriol. 178:3015-3024 (1996)), which naturally catalyzesthe reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzymecan be enhanced by expressing bcd in conjunction with expression of theC. acetobutylicum etfAB genes, which encode an electron transferflavoprotein. An additional candidate for the enoyl-CoA reductase stepis the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeisteret al., J Biol. Chem. 280:4329-4338 (2005)). A construct derived fromthis sequence following the removal of its mitochondrial targetingleader sequence was cloned in E. coli resulting in an active enzyme(Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). This approachis well known to those skilled in the art of expressing eukaryoticgenes, particularly those with leader sequences that may target the geneproduct to a specific intracellular compartment, in prokaryoticorganisms. A close homolog of this gene, TDE0597, from the prokaryoteTreponema denticola represents a third enoyl-CoA reductase which hasbeen cloned and expressed in E. coli (Tucci and Martin, Febs Letters581:1561-1566 (2007)).

GenBank Gene name GI# Accession # Organism bcd 15895968 NP_349317.1Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridiumacetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum TER62287512 Q5EU90.1 Euglena gracilis TDE0597 42526113 NP_971211.1Treponema denticola

Additional enoyl-CoA reductase enzyme candidates are found in organismsthat degrade aromatic compounds. Rhodopseudomonas palustris, a modelorganism for benzoate degradation, has the enzymatic capability todegrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes inthe pim operon, pimC and pimD, bear sequence homology to C.acetobutylicum bcd and are predicted to encode a flavin-containingpimeloyl-CoA dehydrogenase (Harrison and Harwood, Microbiology151:727-736 (2005)). The genome of nitrogen-fixing soybean symbiontBradyrhizobium japonicum also contains a pim operon composed of geneswith high sequence similarity to pimC and pimD of R. palustris (Harrisonand Harwood, Microbiology 151:727-736 (2005)).

GenBank Gene name GI# Accession # Organism pimC 39650632 CAE29155Rhodopseudomonas palustris pimD 39650631 CAE29154 Rhodopseudomonaspalustris pimC 27356102 BAC53083 Bradyrhizobium japonicum pimD 27356101BAC53082 Bradyrhizobium japonicum

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase(EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hinderedtrans-enoyl-CoA substrates. This enzyme participates in branched-chainfatty acid synthesis in the nematode Ascarius suum and is capable ofreducing a variety of linear and branched chain substrates including2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA andpentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Twoisoforms of the enzyme, encoded by genes acad1 and acad, have beencharacterized.

GenBank Gene name GI# Accession # Organism acad1 2407655 AAC48316.1Ascarius suum acad 347404 AAA16096.1 Ascarius suum

1.4.1.a Oxidoreductase (Ketone or Aldehyde to Amino).

Oxidoreductases in the EC class 1.4.1 that convert an aldehyde or ketoneto its corresponding amine group catalyze several biosynthetic steps inthe disclosed pathways. In FIG. 12, the conversions of OHED to 2-AHE(Step J), 2-OHD to 2-AHD (Step H) and adipate semialdehyde to6-aminocaproate (Step E) are catalyzed by OHED aminating oxidoreductase,2-OHD aminating oxidoreductase and adipate semialdehyde aminatingoxidoreductase. In FIG. 13, conversion of 6-aminocaproate semialdehydeto HMDA (Step H) and 6-acetamidohexanal to 6-acetamidohexanamine (StepG), are also catalyzed by aminating oxidoreductases.

Most aminating oxidoreductases catalyze the reversible oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor, and thereactions are typically reversible. Exemplary enzymes include glutamatedehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase(deaminating), encoded by ldh, and aspartate dehydrogenase(deaminating), encoded by nadX. The gdhA gene product from Escherichiacoli (Korber et al., J Mol. Biol. 234:1270-1273 (1993); and McPherson etal., Nucleic Acids Res. 11:5257-5266 (1983)), gdh from Thermotogamaritime (Kort et al., Extremophiles. 1:52-60 (1997); Lebbink et al., JMol. Biol. 280:287-296 (1998); and Lebbink et al., J Mol. Biol.289:357-369 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsbyet al., Gene 349:237-244 (2005)) catalyze the reversible interconversionof glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H),NAD(H), or both, respectively. The ldh gene of Bacillus cereus encodesthe LeuDH protein that has a wide of range of substrates includingleucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula,Biotechnol Bioeng 68:557-562 (2000); and Stoyan et al., J Biotechnol.54:77-80 (1997)). The nadX gene from Thermotoga maritime encoding forthe aspartate dehydrogenase is involved in the biosynthesis of NAD (Yanget al., J Biol. Chem. 278:8804-8808 (2003)).

GenBank Gene name GI# Accession # Organism gdhA 118547 P00370Escherichia coli gdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827NP_279651.1 Halobacterium salinarum ldh 61222614 P0A393 Bacillus cereusnadX 15644391 NP_229443.1 Thermotoga maritima

Lysine 6-dehydrogenase (deaminating), encoded by lysDH, catalyzes theoxidative deamination of the 6-amino group of L-lysine to form2-aminoadipate-6-semialdehyde, which in turn non-enzymatically cyclizesto form A′-piperideine-6-carboxylate (Misono and Nagasaki, J. Bacteriol.150:398-401 (1982)). Exemplary enzymes can be found in Geobacillusstearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942(2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem.106:76-80 (1989); and Misono and Nagasaki, J. Bacteriol. 150:398-401(1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB.Rep. 41:790-795 (2008)). Such enzymes are particularly good candidatesfor converting adipate semialdehyde to 6-aminocaproate given thestructural similarity between adipate semialdehyde and2-aminoadipate-6-semialdehyde.

GenBank Gene name GI# Accession # Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

2.3.1.a Acyltransferase (Transferring CoA to Phospho).

Acyltransferases that exchange a CoA moiety for a phosphate are in theEC class 2.3.1. Transformations in this category include the conversionsof 6-AAHOP to 6-acetamidohexanoyl-CoA (FIG. 13, Step K) and 6-AHOP to6-aminocaproyl-CoA (FIG. 13, Step L). Exemplary phosphate-transferringacyltransferases include phosphotransacetylase (EC 2.3.1.8), encoded bypta, and phosphotransbutyrylase (EC 2.3.1.19), encoded by ptb. The ptagene from E. coli encodes an enzyme that reversibly converts acetyl-CoAinto acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569(1969)). This enzyme can also utilize propionyl-CoA as a substrate,forming propionate in the process (Hesslinger et al., Mol. Microbiol27:477-492 (1998)). Similarly, the ptb gene from C. acetobutylicumencodes phosphate transbutyrylase, an enzyme that reversibly convertsbutyryl-CoA into butyryl-phosphate (Walter et al., Gene 134:107-111(1993); and Wiesenborn et al., Appl Environ. Microbiol 55:317-322(1989)). Additional ptb genes are found in butyrate-producing bacteriumL2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al., Curr. Microbiol 42:345-349 (2001)).

GenBank Gene name GI# Accession # Organism Pta 16130232 NP_416800.1Escherichia coli Ptb 15896327 NP_349676 Clostridium acetobutylicum Ptb38425288 AAR19757.1 butyrate-producing bacterium L2-50 Ptb 10046659CAC07932.1 Bacillus megaterium

2.3.1.c Acyltransferase (N-acetyltransferase).

N-Acetyltransferases transfer an acetyl group to an amine, forming anN-acetyl group. N-Acetylation serves diverse functions in biologicalsystems including transcriptional regulation, nuclear import, chromosomeassembly and nucleosome remodeling (Kouzarides, EMBO J 19:1176-1179(2000)). N-Acetylation of metabolic intermediates of argininebiosynthetic pathways serves both to protect reactive intermediates fromspontaneous cyclization and also to sequester pathway intermediates fromcompeting pathways (Caldovic and Tuchman, Biochem. J 372:279-290(2003)). Acetylation of 6-ACA (FIG. 13, step D) serves a similar role inthe proposed HMDA biosynthesis route of FIG. 13, protecting reactiveintermediates from spontaneous cyclization.

One candidate enzyme for acetylating 6-ACA is lysine N-acetyltransferase(EC 2.3.1.32), an enzyme which selectively transfers the acetyl moietyfrom acetyl phosphate to the terminal amino group of L-lysine,beta-L-lysine or L-ornithine. Although this enzyme is not known toacetylate 6-ACA, this substrate is structurally similar to the naturalsubstrate. Lysine N-acetyltransferase has been characterized in Bostaurus (Paik. and Kim, Arch. Biochem. Biophys. 108:221-229, 1964) andMethanosarcina mazei (Pfluger et al., Appl Environ. Microbiol69:6047-6055 (2003)). Methanogenic archaea M. maripaludis, M.acetivorans, M. barkeri and M. jannaschii are also predicted to encodeenzymes with this functionality (Pfluger et al., Appl Environ. Microbiol69:6047-6055 (2003)).

GenBank Gene name GI# Accession # Organism ablB 21227037 NP_632959.1Methanosarcina mazei yodP 44921183 CAF30418 Methanococcus maripaludisMA3978 20092772 NP_618847.1 Methanosarcina acetivorans MJ0635 15668816NP_247619.1 Methanocaldococcus jannaschii Mbar_A0671 73668215YP_304230.1 Methanosarcina barkeri

Alternately, 6-ACA acetylation can be catalyzed by an enzyme in the GNATfamily of N-acetyltransferases. Such enzymes transfer an acetyl groupfrom acetyl-CoA to a primary amine. The enzyme spermidineN-acetyltransferase (SSAT), also known as diamine N-acetyltransferase(EC 2.3.1.57), is capable of acetylating a variety of small moleculesubstrates. Purified enzymes from Ascaris suum and Onchocerca volvulusexhibit a broad substrate range that includes HMDA (Davids et al., Mol.Biochem. Parasitol. 64:341-344 (1994); and Wittich and Walter, Mol.Biochem. Parasitol. 38:13-17 (1990)), but the associated genes have notbeen identified to date. Other enzymes with this functionality are foundin Bacillus subtilis (Forouhar et al., J Biol. Chem. 280:40328-40336(2005)) and Homo sapiens (Casero and Pegg, FASEB J 7:653-661 (1993)). Aclosely related enzyme is thialysine N-acetyltransferase in C. elegans,an enzyme that accepts a range of substrates including lysine,ornithine, thialysine and others (bo-Dalo et al., Biochem. J 384:129-137(2004)). Amino acid residues involved in substrate binding wereidentified in the thialysine N-acetyltransferase from Leishmania major(Luersen, K., FEBS Lett. 579:5347-5352 (2005)). An additional candidateis the diaminobutyrate acetyltransferase (EC 2.3.1.178), an enzymeparticipating in ectoine biosynthesis in Methylomicrobium alcaliphilum(Reshetnikov et al., Arch. Microbiol 184:286-297 (2006)) C. salexigens(formerly Halomonas elongata) (Canovas et al., Syst. Appl Microbiol21:487-497 (1998)).

GenBank Gene name GI# Accession # Organism paiA 16080268 NP_391095.1Bacillus subtilis SSAT1 114322 P21673 Homo sapiens D2023.4 17559148NP_505978.1 Caenorhabditis elegans LmjF36.2750 68129928 CAJ09234.1Leishmania major ectA 68366269 AAY96770.1 Methylomicrobium alcaliphilum20Z ectA 6685422 Q9ZEU8.1 Chromohalobacter salexigens

An additional enzyme candidate for acetylating 6-ACA (FIG. 13, Step D)and de-acetylating 6-acetamidehexanamine (FIG. 13, Step H) is ornithineacetyltransferase (OAT, EC 2.3.1.35 and EC 2.3.1.1), a bifunctionalenzyme which catalyzes two steps of arginine biosynthesis (FIG. 14A).The first step of arginine biosynthesis (FIG. 14A, step 1) is theN-acetylation of glutamate, catalyzed by OAT with acetyl-CoA as anacetyl donor (O'Reilly and Devine, Microbiology 140 (Pt 5):1023-1025(1994)). OAT also catalyzes the fifth step of arginine biosynthesis(FIG. 14A, step 2), in which an N-acetyl group is transferred fromN-acetyl-L-ornithine to L-glutamate, the first metabolite in thearginine biosynthesis pathway. This transformation serves to recycle theacetyl group and regenerate N-acetylglutamate, conserving energy andthereby making the linear pathway a cyclic route. A similar strategy canbe employed in HMDA biosynthesis from 6-aminocaproate, with a singleenzyme acetylating 6-aminocaproate and de-acetylating6-acetamidohexanamine to form HMDA (FIG. 14B). Exemplary OAT enzymes areencoded by argJ in Bacillus subtilis (O'Reilly and Devine, Microbiology140 (Pt 5):1023-1025 (1994); and Sakanyan et al., Journal of GeneralMicrobiology 138:125-130 (1992)) and ECM40 in S. cerevisiae (Abadjievaet al., J Biol. Chem. 275:11361-11367 (2000); and Liu et al., Eur. JBiochem. 228:291-296 (1995)). Crystal structures of the enzymes fromyeast (Maes et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst.Commun. 62:1294-1297 (2006)) and Mycobacterium tuberculosis(Sankaranarayanan et al., Acta Crystallogr. Sect. F. Struct. Biol.Cryst. Commun. 65:173-176 (2009)) are available. Although encoded by asingle open reading frame, OAT enzymes have distinct alpha and betasubunit peptides (Liu et al., Eur. J Biochem. 228:291-296 (1995)).

GenBank Gene name GI# Accession # Organism argJ 16078185 NP_389002.1Bacillus subtilis ECM40 (ARG7) 6323707 NP_013778.1 Saccharomycescerevisiae Rv1653 15608791 NP_216169.1 Mycobacterium tuberculosis

2.3.1.d Acyltransferase (Formate C-Acyltransferase).

The acylation of ketoacids HODH, OHED and 2-OHD to their correspondingCoA derivatives (FIG. 12, Steps L, P and Q) and concurrent release offormate, is catalyzed by formate C-acyltransferase enzymes in the ECclass 2.3.1. Enzymes in this class include pyruvate formate-lyase andketoacid formate-lyase. Pyruvate formate-lyase (PFL, EC 2.3.1.54),encoded by pflB in E. coli, converts pyruvate into acetyl-CoA andformate. The active site of PFL contains a catalytically essentialglycyl radical that is posttranslationally activated under anaerobicconditions by PFL-activating enzyme (PFL-AE, EC 1.97.1.4) encoded bypflA (Knappe et al., Proc. Natl. Acad. Sci U.S.A 81:1332-1335 (1984);and Wong et al., Biochemistry 32:14102-14110 (1993)). A pyruvateformate-lyase from Archaeglubus fulgidus encoded by pflD has beencloned, expressed in E. coli and characterized (Lehtio, L. and A.Goldman, Protein Eng Des Sel 17:545-552 (2004)). The crystal structuresof the A. fulgidus and E. coli enzymes have been resolved (Lehtio etal., J Mol. Biol. 357:221-235 (2006)). Additional PFL and PFL-AEcandidates are found in Clostridium pasteurianum (Weidner and Sawers, JBacteriol. 178:2440-2444 (1996)) and the eukaryotic alga Chlamydomonasreinhardtii (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)).Keto-acid formate-lyase (EC 2.3.1.-), also known as 2-ketobutyrateformate-lyase (KFL) and pyruvate formate-lyase 4, is the gene product oftdcE in E. coli. This enzyme catalyzes the conversion of 2-ketobutyrateto propionyl-CoA and formate during anaerobic threonine degradation, andcan also substitute for pyruvate formate-lyase in anaerobic catabolism(Simanshu et al., J Biosci. 32:1195-1206 (2007)). The enzyme isoxygen-sensitive and, like PflB, requires post-translationalmodification by PFL-AE to activate a glycyl radical in the active site(Hesslinger et al., Mol. Microbiol 27:477-492 (1998)).

GenBank Gene name GI# Accession # Organism pflB 16128870 NP_415423.1Escherichia coli pflA 16128869 NP_415422.1 Escherichia coli tdcE48994926 AAT48170.1 Escherichia coli pflD 11499044 NP_070278.1Archaeglubus fulgidus pfl 2500058 Q46266.1 Clostridium pasteurianum act1072362 CAA63749.1 Clostridium pasteurianum pfl1 159462978XP_001689719.1 Chlamydomonas reinhardtii pflA1 159485246 XP_001700657.1Chlamydomonas reinhardtii

2.6.1.a Aminotransferase.

Steps E, H and J of FIG. 12 and Steps C and G of FIG. 13 requireconversion of an aldehyde or ketone to an amino group. Thistransformation can be accomplished by an aminotransferase (EC 2.6.1.-).The conversion of an aldehyde to a terminal amine (FIG. 12, Step E; FIG.13, Steps C and G) can be catalyzed by gamma-aminobutyrate transaminase(GABA transaminase). One E. coli GABA transaminase is encoded by gabTand transfers an amino group from glutamate to the terminal aldehyde ofsuccinic semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042(1990)). This enzyme exhibits a broad substrate range (Liu et al.,Biochemistry 43:10896-10905 (2004)). The gene product of puuE encodesthe other 4-aminobutyrate transaminase in E. coli (Kurihara et al., JBiol. Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus,Pseudomonas fluorescens, and Sus scrofa have been shown to react with6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); andScott and Jakoby, J Biol. Chem. 234:932-936 (1959)).

GenBank Gene name GI# Accession # Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. Such enzymes are particularly wellsuited for carrying out the conversion of 6-aminocaproate semialdehydeto HMDA. The E. coli putrescine aminotransferase is encoded by the ygjGgene and the purified enzyme also was able to transaminate cadaverineand spermidine (Samsonova et al., BMC. Microbiol 3:2 (2003)). Inaddition, activity of this enzyme on 1,7-diaminoheptane and with aminoacceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) hasbeen reported (Kim, J Biol. Chem. 239:783-786 (1964); and Samsonova etal., BMC. Microbiol 3:2 (2003)). A putrescine aminotransferase withhigher activity with pyruvate as the amino acceptor thanalpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu etal., J. Bacteriol. 184:3765-3773 (2002)).

GenBank Gene name GI# Accession # Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa

Additional candidate enzymes include beta-alanine/alpha-ketoglutarateaminotransferases which produce malonic semialdehyde from beta-alanine(WO08027742). The gene product of SkPYD4 in Saccharomyces kluyveri wasshown to preferentially use beta-alanine as the amino group donor(Andersen and Hansen, Gene 124:105-109 (1993)). SkUGA1 encodes ahomologue of Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramoset al., Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes anenzyme involved in both β-alanine and GABA transamination (Andersen andHansen, Gene 124:105-109 (1993)). 3-Amino-2-methylpropionatetransaminase catalyzes the transformation from methylmalonatesemialdehyde to 3-amino-2-methylpropionate. The enzyme has beencharacterized in Rattus norvegicus and Sus scrofa and is encoded by Abat1968 (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); andTamaki et al., Methods Enzymol. 324:376-389 (2000)).

GenBank Gene name GI# Accession # Organism SkyPYD4 98626772 ABF58893.1Saccharomyces kluyveri SkUGA1 98626792 ABF58894.1 Saccharomyces kluyveriUGA1 6321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191P50554.3 Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

Steps J and H of FIG. 12 are catalyzed by aminotransferases thattransform amino acids into oxo-acids. In Step J, OHED is transaminatedto form 2-AHE by OHED aminotransferase. The transamination of 2-OHD to2-AHD by 2-OHD aminotransferase (Step H) is a similar reaction. Anexemplary enzyme candidate for catalyzing these reactions is aspartateaminotransferase, an enzyme that naturally transfers an oxo group fromoxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate.Aspartate is similar in structure to OHED and 2-AHD. Aspartateaminotransferase activity is catalyzed by, for example, the geneproducts of aspC from Escherichia coli (Yagi et al., FEBS Lett.100:81-84, (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)),AAT2 from Saccharomyces cerevisiae (Yagi et al., J Biochem. 92:35-43(1982)) and ASPS from Arabidopsis thaliana (de la Torre et al., Plant J46:414-425 (2006); Kwok and Hanson, J Exp. Bot. 55:595-604 (2004); andWilkie and Warren, Protein Expr. Purif. 12:381-389 (1998)). The enzymefrom Rattus norvegicus has been shown to transaminate alternatesubstrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid(Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferasesthat work on other amino-acid substrates can catalyze thistransformation. Valine aminotransferase catalyzes the conversion ofvaline and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene,avtA, encodes one such enzyme (Whalen and Berg, C. J. Bacteriol.150:739-746 (1982)). This gene product also catalyzes the transaminationof α-ketobutyrate to generate α-aminobutyrate, although the amine donorin this reaction has not been identified (Whalen and Berg, J Bacteriol.158:571-574 (1984)). The gene product of the E. coli serC catalyzes tworeactions, phosphoserine aminotransferase and phosphohydroxythreonineaminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)),and activity on non-phosphorylated substrates could not be detected(Drewke et al., FEBS. Lett. 390:179-182 (1996)).

GenBank Gene name GI# Accession # Organism aspC 16128895 NP_415448.1Escherichia coli AAT2 1703040 P23542.3 Saccharomyces cerevisiae ASP520532373 P46248.2 Arabidopsis thaliana Got2 112987 P00507 Rattusnorvegicus avtA 49176374 YP_026231.1 Escherichia coli serC 16128874NP_415427.1 Escherichia coli

2.7.2.a Phosphotransferase (Carboxy Acceptor).

Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylicacids to phosphonic acids with concurrent hydrolysis of one ATP. Steps Aand E in FIG. 13 require a phosphotransferase to activate the carboxylgroups of 6-ACA (Step A) and 6-acetamidohexanoate (Step E) to theircorresponding phosphonic acids. Butyrate kinase carries out thereversible conversion of butyryl-phosphate to butyrate duringacidogenesis in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol56:1576-1583 (1990)). This enzyme is encoded by either of the two bukgene products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38(2000)). Related enzyme isobutyrate kinase from Thermotoga maritima hasalso been expressed in E. coli and crystallized (Diao et al., ActaCrystallogr. D. Biol. Crystallogr. 59:1100-1102 (2003); and Diao andHasson, J. Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes theATP-dependent phosphorylation of aspartate and participates in thesynthesis of several amino acids. The aspartokinase III enzyme in E.coli, encoded by lysC, has a broad substrate range and the catalyticresidues involved in substrate specificity have been elucidated (Kengand Viola, Arch. Biochem. Biophys. 335:73-81 (1996)). Two additionalkinases in E. coli are also good candidates: acetate kinase andgamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein, J. Biol. Chem. 251:6775-6783 (1976)),phosphorylates propionate in addition to acetate (Hesslinger et al.,Mol. Microbiol 27:477-492 (1998)). The E. coli gamma-glutamyl kinase,encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)),phosphorylates the gamma carbonic acid group of glutamate.

GenBank Gene name GI# Accession # Organism buk1 15896326 NP_349675Clostridium acetobutylicum buk2 20137415 Q97II1 Clostridiumacetobutylicum buk2 6685256 Q9X278.1 Thermotoga maritima lysC 16131850NP_418448.1 Escherichia coli ackA 16130231 NP_416799.1 Escherichia coliproB 16128228 NP_414777.1 Escherichia coli

Acetylglutamate kinase phosphorylates acetylated glutamate duringarginine biosynthesis and is a good candidate for phosphorylating6-acetamidohexanoate (FIG. 13, Step E). This enzyme is not known toaccept alternate substrates; however, several residues of the E. colienzyme involved in substrate binding and phosphorylation have beenelucidated by site-directed mutagenesis (Marco-Martin et al., J Mol.Biol. 334:459-476 (2003); and Ramon-Maiques et al., Structure.10:329-342 (2002)). The enzyme is encoded by argB in Bacillus subtilisand E. coli (Parsot et al., Gene 68:275-283 (1988)), and ARG5,6 in S.cerevisiae (Pauwels et al., Eur. J Biochem. 270:1014-1024 (2003)). TheARG5,6 gene of S. cerevisiae encodes a polyprotein precursor that ismatured in the mitochondrial matrix to become acetylglutamate kinase andacetylglutamylphosphate reductase, an enzyme candidate for the reductionof 6-AAHOP (FIG. 13, Step F).

GenBank Gene name GI# Accession # Organism argB 145698337 NP_418394.3Escherichia coli argB 16078186 NP_389003.1 Bacillus subtilis ARG5, 66320913 NP_010992.1 Saccharomyces cerevisiae

2.8.3.a Coenzyme-A Transferase.

Coenzyme-A (CoA) transferases catalyze the reversible transfer of a CoAmoiety from one molecule to another. In Step M of FIG. 13,3-aminocaproyl-CoA is formed by the transfer of a CoA group fromacetyl-CoA, succinyl-CoA, or another CoA donor. A similar transformationis catalyzed by 6-acetamidohexanoate CoA-transferase, shown in Step I ofFIG. 13. Exemplary CoA transferase candidates are catalyzed by the geneproducts of cat1, cat2, and cat3 of Clostridium kluyveri which have beenshown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoAtransferase activity, respectively (Seedorf et al., Proc. Natl. Acad.Sci U.S.A 105:2128-2133 (2008); and Sohling and Gottschalk, J.Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities arealso present in Trichomonas vaginalis (van Grinsven et al., J. Biol.Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J.Biol. Chem. 279:45337-45346 (2004)).

GenBank Gene name GI# Accession # Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3146349050 EDK35586.1 Clostridium kluyveri TVAG_395550 123975034XP_001330176 Trichomonas vaginalis G3 Tb11.02.0290 71754875 XP_828352Trypanosoma brucei

A CoA transferase that can utilize acetyl-CoA as the CoA donor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr. D.Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al.,Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has abroad substrate range (Sramek and Frerman, Arch. Biochem. Biophys.171:14-26 (1975)) and has been shown to transfer the CoA moiety toacetate from a variety of branched and linear acyl-CoA substrates,including isobutyrate (Matthies and Schink, Appl Environ. Microbiol58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys.Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al.,Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme isinduced at the transcriptional level by acetoacetate, so modification ofregulatory control may be necessary for engineering this enzyme into apathway (Pauli and Overath, Eur. J Biochem. 29:553-562 (1972)). Similarenzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al.,Appl. Environ. Microbiol 68:5186-5190 (2002)), Clostridiumacetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583(1990); and Wiesenborn et al., Appl. Environ. Microbiol 55:323-329(1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci. Biotechnol Biochem. 71:58-68 (2007)).

GenBank Gene name GI# Accession # Organism AtoA 2492994 NP_416726Escherichia coli K12 AtoD 2492990 NP_416725 Escherichia coli K12 actA62391407 YP_226809.1 Corynebacterium glutamicum ATCC 13032 cg059262389399 YP_224801.1 Corynebacterium glutamicum ATCC 13032 ctfA 15004866NP_149326.1 Clostridium acetobutylicum ctfB 15004867 NP_149327.1Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridiumsaccharoperbutylacetonicum

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and3-butenoyl-CoA (Mack et al., Eur. Biochem. 226:41-51 (1994)). The genesencoding this enzyme are gctA and gctB. This enzyme has reduced butdetectable activity with other CoA derivatives including glutaryl-CoA,2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. JBiochem. 118:315-321 (1981)). The enzyme has been cloned and expressedin E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

GenBank Gene name GI# Accession # Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

Yet another CoA transferase is the two-unit succinyl-CoA:3:oxoacid-CoAtransferase encoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek etal., J. Bacteriol. 184:207-215 (2002)). Similar enzymes based onhomology exist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene146:23-30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid-CoAtransferases are present in Helicobacter pylori (Corthesy-Theulaz etal., J Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stolset al., Protein Expr. Purif. 53:396-403 (2007)).

GenBank Gene name GI# Accession # Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

3.5.1.a Hydrolase (Acting on Linear Amides).

Deacetylation of linear acetamides is catalyzed by an amidohydrolase inthe 3.5.1 family of enzymes. Such an enzyme is required for thedeacetylation of 6-acetamidohexanamine to HMDA (FIG. 13, Step H). Anenzyme catalyzing a similar transformation is 4-acetamidobutyratedeacetylase (EC 3.5.1.63), which naturally deacetylates4-acetamidobutyrate. The enzyme, studied for its role in putrescinedegradation in Candida boidinii (Gillyon et al., Journal of GeneralMicrobiology 133:2477-2485 (1987)), has been shown to deacetylate avariety of substrates including 6-acetamidohexanoate (Haywood and Large,Journal of General Microbiology 132:7-14 (1986)). Although6-Acetamidohexanoate is similar in structure to the desired substrate,deacetylation of this compound (FIG. 13, step D, reverse reaction) mayhinder efficient production of HMDA. Protein engineering or directedevolution may be required to improve specificity for6-acetamidohexanamine. The gene associated with this activity has notbeen identified to date.

Acetylpolyamine amidohydrolase (EC 3.5.1.62), is another candidateenzyme that forms the diamines putrescine and cadaverine from theiracetylated precursors. The acetylpolyamine deacetylase (AphA) fromMycoplana ramosa has been cloned in E. coli and characterized (Sakuradaet al., J Bacteriol. 178:5781-5786 (1996)) and a crystal structure isavailable (Fujishiro et al., Biochem. Biophys. Res. Commun.157:1169-1174 (1988)). This enzyme has also been studied in Micrococcusluteus, but the associated gene has not been identified to date (Suzukiet al., Biochim. Biophys. Acta 882:140-142 (1986)). A protein thehistone deacetylase superfamily with high sequence similarity to AphAwas identified in the M. luteus genome (evalue=1e-18, 37% identity). TheN-acetyl-L-ornithine deacetylase from E. coli is another candidateamidohydrolase (EC 3.5.1.16). The E. coli enzyme, encoded by the argEgene (McGregor et al., J Am. Chem. Soc. 127:14100-14107 (2005); andMeinnel et al., Bacteriol. 174:2323-2331 (1992)), removes N-acetylgroups from a variety of substrates including ornithine, lysine,glutamine, and other amino acids (Javid-Majd and Blanchard, Biochemistry39:1285-1293 (2000)).

GenBank Gene name GI# Accession # Organism aphA 3023317 Q48935.1Mycoplana ramose MlutDRAFT_1143 172071524 EDT57566.1 Micrococcus luteusargE 16131795 NP_418392.1 Escherichia coli

4.1.1.a Carboxy-Lyase.

Steps D and F in FIG. 12 are catalyzed by 2-ketoacid decarboxylaseenzymes that generate 6-OHE and adipate semialdehyde from OHED (Step F)and 2-OHD (Step D). In addition, alpha-ketoglutarate is decarboxylatedto form pathway precursor succinic semialdehyde by alpha-ketoglutaratedecarboxylase, a keto-acid decarboxylase. The decarboxylation ofketo-acids is catalyzed by a variety of enzymes with varied substratespecificities, including pyruvate decarboxylase (EC 4.1.1.1),benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutaratedecarboxylase and branched-chain alpha-ketoacid decarboxylase. Pyruvatedecarboxylase (PDC), also termed keto-acid decarboxylase, is a keyenzyme in alcoholic fermentation, catalyzing the decarboxylation ofpyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has abroad substrate range for aliphatic 2-keto acids including2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate(22). This enzyme has been extensively studied, engineered for alteredactivity, and functionally expressed in E. coli (Killenberg-Jabs et al.,Eur. J. Biochem. 268:1698-1704 (2001); Li, H. and F. Jordan,Biochemistry. 38:10004-10012 (1999); and ter Schure et al., Appl.Environ. Microbiol. 64:1303-1307 (1998)). The PDC from Zymomonasmobilus, encoded by pdc, also has a broad substrate range and has been asubject of directed engineering studies to alter the affinity fordifferent substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The crystal structure of this enzyme is available(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Otherwell-characterized PDC candidates include the enzymes from Acetobacterpasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) andKluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263(2002)).

GenBank Gene name GI# Accession # Organism pdc 118391 P06672.1 Zymomonasmobilus pdc1 30923172 P06169 Saccharomyces cerevisiae pdc 20385191Q8L388 Acetobacter pasteurians pdc1 52788279 Q12629 Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.,Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry42:1820-1830 (2003)). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity (Km)of naturally and non-naturally occurring substrates (Siegert et al.,Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzymehave been further modified by directed engineering (Lingen et al.,Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726(2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, hasalso been characterized experimentally (Barrowman et al., FEMSMicrobiology Letters 34:57-60 (1986)). Additional gene candidates fromPseudomonas stutzeri, Pseudomonas fluorescens and other organisms can beinferred by sequence homology or identified using a growth selectionsystem developed in Pseudomonas putida (Henning et al., Appl. Environ.Microbiol. 72:7510-7517 (2006)).

GenBank Gene name GI# Accession # Organism mdlC 3915757 P20906.2Pseudomonas putida mdlC 81539678 Q9HUR2.1 Pseudomonas aeruginosa dpgB126202187 ABN80423.1 Pseudomonas stutzeri ilvB-1 70730840 YP_260581.1Pseudomonas fluorescens

A third enzyme capable of decarboxylating 2-oxoacids isalpha-ketoglutarate decarboxylase (KGD). The substrate range of thisclass of enzymes has not been studied to date. The KDC fromMycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci U S. A102:10670-10675 (2005)) has been cloned and functionally expressed inother internal projects at Genomatica. However, it is not an idealcandidate for strain engineering because it is large (˜130 kD) andGC-rich. KDC enzyme activity has been detected in several species ofrhizobia including Bradyrhizobium japonicum and Mesorhizobium loti(Green et al., J Bacteriol. 182:2838-2844 (2000)). Although theKDC-encoding gene(s) have not been isolated in these organisms, thegenome sequences are available and several genes in each genome areannotated as putative KDCs. A KDC from Euglena gracilis has also beencharacterized but the gene associated with this activity has not beenidentified to date (Shigeoka and Nakano, Arch. Biochem. Biophys.288:22-28 (1991)). The first twenty amino acids starting from theN-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 1) (Shigeokaand Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene can beidentified by testing candidate genes containing this N-terminalsequence for KDC activity.

GenBank Gene name GI# Accession # Organism kgd 160395583 O50463.4Mycobacterium tuberculosis kgd 27375563 NP_767092.1 Bradyrhizobiumjaponicum kgd 13473636 NP_105204.1 Mesorhizobium loti

A fourth candidate enzyme for catalyzing this step is branched chainalpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shownto act on a variety of compounds varying in chain length from 3 to 6carbons (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988); and Smitet al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme inLactococcus lactis has been characterized on a variety of branched andlinear substrates including 2-oxobutanoate, 2-oxohexanoate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate andisocaproate (Smit et al., Appl Environ Microbiol. 71:303-311 (2005)).The enzyme has been structurally characterized (Berg et al., Science.318:1782-1786 (2007)). Sequence alignments between the Lactococcuslactis enzyme and the pyruvate decarboxylase of Zymomonas mobilusindicate that the catalytic and substrate recognition residues arenearly identical (Siegert et al., Protein Eng Des Sel 18:345-357(2005)), so this enzyme would be a promising candidate for directedengineering. Decarboxylation of alpha-ketoglutarate by a BCKA wasdetected in Bacillus subtilis; however, this activity was low (5%)relative to activity on other branched-chain substrates (Oku and Kaneda,J Biol Chem. 263:18386-18396 (1988)) and the gene encoding this enzymehas not been identified to date. Additional BCKA gene candidates can beidentified by homology to the Lactococcus lactis protein sequence. Manyof the high-scoring BLASTp hits to this enzyme are annotated asindolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvatedecarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation ofindolepyruvate to indoleacetaldehyde in plants and plant bacteria.

GenBank Gene name GI# Accession # Organism kdcA 44921617 AAS49166.1Lactococcus lactis

Recombinant branched chain alpha-keto acid decarboxylase enzymes derivedfrom the E1 subunits of the mitochondrial branched-chain keto aciddehydrogenase complex from Homo sapiens and Bos taurus have been clonedand functionally expressed in E. coli (Davie et al., J. Biol. Chem.267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). Inthese studies, the authors found that co-expression of chaperonins GroELand GroES enhanced the specific activity of the decarboxylase by500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). Theseenzymes are composed of two alpha and two beta subunits.

GenBank Gene name GI# Accession # Organism BCKDHB 34101272 NP_898871.1Homo sapiens BCKDHA 11386135 NP_000700.1 Homo sapiens BCKDHB 115502434P21839 Bos taurus BCKDHA 129030 P11178 Bos taurus

The decarboxylation of 2-AHD to 6-aminocaproate (FIG. 12, Step I) iscatalyzed by an amino acid decarboxylase such as aspartatedecarboxylase. Aspartate decarboxylase participates in pantothenatebiosynthesis and is encoded by gene panD in Escherichia coli (Dusch etal., Appl. Environ. Microbiol 65:1530-1539 (1999); Merke and Nichols,FEMS Microbiol Lett. 143:247-252 (1996); Ramjee et al., Biochem. J 323(Pt 3):661-669 (1997); and Schmitzberger et al., EMBO J 22:6193-6204(2003)). Similar enzymes from Mycobacterium tuberculosis (Chopra et al.,Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutamicum(Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999)) have beenexpressed and characterized in E. coli.

GenBank Gene name GI# Accession # Organism panD 67470411 P0A790Escherichia coli K12 panD 18203593 Q9X4N0 Corynebacterium glutamicumpanD 54041701 P65660.1 Mycobacterium tuberculosis

4.1.2.a Aldehyde-Lyase.

HOHD aldolase, also known as HHED aldolase, catalyzes the conversion of4-hydroxy-2-oxo-heptane-1,7-dioate (HOHD) into pyruvate and succinicsemialdehyde (FIG. 12, Step A). The enzyme is a divalent metal iondependent class II aldolase, catalyzing the final step of4-hydroxyphenylacetic acid degradation in E. coli C, E. coli W, andother organisms. In the native context, the enzyme functions in thedegradative direction. The reverse (condensation) reaction isthermodynamically unfavorable; however the equilibrium can be shiftedthrough coupling HOHD aldolase with downstream pathway enzymes that workefficiently on reaction products. Such strategies have been effectivefor shifting the equilibrium of other aldolases in the condensationdirection (Nagata et al., Appl Microbiol Biotechnol 44:432-438 (1995);and Pollard et al., Appl Environ. Microbiol 64:4093-4094 (1998)). The E.coli C enzyme, encoded by hpcH, has been extensively studied and hasrecently been crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007);and Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme isencoded by hpaI (Prieto et al., J Bacteriol. 178:111-120 (1996)).

GenBank Gene name GI# Accession # Organism hpcH 633197 CAA87759.1Escherichia coli C hpaI 38112625 AAR11360.1 Escherichia coli W

4.2.1.a Hydro-Lyase.

The enzyme OHED hydratase participates in 4-hydroxyphenylacetic aciddegradation, where it converts 2-oxo-hept-4-ene-1,7-dioate (OHED) to2-oxo-4-hydroxy-hepta-1,7-dioate (HODH) using magnesium as a cofactor(Burks et al., Am. Chem. Soc. 120 (1998)) (FIG. 12, Step B). OHEDhydratase enzyme candidates have been identified and characterized in E.coli C (Izumi et al., J Mol. Biol. 370:899-911 (2007); and Roper et al.,Gene 156:47-51 (1995)) and E. coli W (Prieto et al., J Bacteriol.178:111-120 (1996)). Sequence comparison reveals homologs in a range ofbacteria, plants and animals. Enzymes with highly similar sequences arecontained in Klebsiella pneumonia (91% identity, evalue=2e-138) andSalmonella enterica (91% identity, evalue=4e-138), among others.

GenBank Gene name GI# Accession # Organism hpcG 556840 CAA57202.1Escherichia coli C hpaH 757830 CAA86044.1 Escherichia coli W hpaH150958100 ABR80130.1 Klebsiella pneumoniae Sari_01896 160865156ABX21779.1 Salmonella enterica

Dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (FIG. 12,Step M) is catalyzed by an enzyme with enoyl-CoA hydratase activity.3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase,dehydrates 3-hydroxyisobutyryl-CoA to form crotonoyl-CoA (FIG. 14, step2). Crotonase enzymes are required for n-butanol formation in someorganisms, particularly Clostridial species, and also comprise one stepof the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilicArchaea of the genera Sulfolobus, Acidianus, and Metallosphaera.Exemplary genes encoding crotonase enzymes can be found in C.acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boyntonet al., J. Bacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmer andGottschalk, FEBS Lett. 21:351-354 (1972)), and Metallosphaera sedula(Berg et al., Science. 318:1782-1786 (2007)) though the sequence of thelatter gene is not known.

GenBank Gene name GI# Accession # Organism crt 15895969 NP_349318.1Clostridium acetobutylicum crt1 153953091 YP_001393856.1 Clostridiumkluyveri

Enoyl-CoA hydratases (EC 4.2.1.17) also catalyze the dehydration of3-hydroxyacyl-CoA substrates (Agnihotri and Liu., J. Bacteriol.188:8551-8559(2003); Conrad et al., J. Bacteriol. 118:103-111 (1974);and Roberts et al., Arch. Microbiol 117:99-108 (1978)). The enoyl-CoAhydratase of Pseudomonas putida, encoded by ech, catalyzes theconversion of 3-hydroxybutyryl-CoA to crotonoyl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratasecandidates are phaA and phaB, of P. putida, and paaA and paaB from P.fluorescens (Olivera et al., Proc. Natl. Acad. Sci U.S.A 95:6419-6424(1998)). The gene product of pimF in Rhodopseudomonas palustris ispredicted to encode an enoyl-CoA hydratase that participates inpimeloyl-CoA degradation (Harrison and Harwood, Microbiology 151:727-736(2005)). Lastly, a number of Escherichia coli genes have been shown todemonstrate enoyl-CoA hydratase functionality including maoC (Park andLee, J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., J Biochem.270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686(2004)) and paaG (Ismail et al., J Biochem. 270:3047-3054(2003); Parkand Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park andYup, Biotechnol Bioeng 86:681-686 (2004)).

GenBank Gene name GI# Accession # Organism ech 26990073 NP_745498.1Pseudomonas putida paaA 26990002 NP_745427.1 Pseudomonas putida paaB26990001 NP_745426.1 Pseudomonas putida phaA 106636093 ABF82233.1Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonasfluorescens pimF 39650635 CAE29158 Rhodopseudomonas palustris maoC16129348 NP_415905.1 Escherichia coli paaF 16129354 NP_415911.1Escherichia coli paaG 16129355 NP_415912.1 Escherichia coli

Alternatively, the E. coli gene products of fadA and fadB encode amultienzyme complex involved in fatty acid oxidation that exhibitsenoyl-CoA hydratase activity (Nakahigashi and Inokuchi, Nucleic AcidsRes. 18:4937 (1990); Yang, J. Bacteriol. 173:7405-7406 (1991); and Yanget al., Biochemistry 30:6788-6795 (1991)). Knocking out a negativeregulator encoded by fadR can be utilized to activate the fadB geneproduct (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI andfadJ genes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805(2003)).

GenBank Gene name GI# Accession # Organism fadA 49176430 YP_026272.1Escherichia coli fadB 16131692 NP_418288.1 Escherichia coli fadI16130275 NP_416844.1 Escherichia coli fadJ 16130274 NP_416843.1Escherichia coli fadR 16129150 NP_415705.1 Escherichia coli

6.2.1.a Acid-Thiol Ligase (Also Called CoA Synthetase).

Steps I and M of FIG. 13 require acid-thiol ligase or CoA synthetasefunctionality to transform 6-ACA and 6-acetamidohexanoate into theircorresponding CoA derivatives (the terms ligase, synthetase, andsynthase are used herein interchangeably and refer to the same enzymeclass). Enzymes catalyzing these exact transformations have not beencharacterized to date; however, several enzymes with broad substratespecificities have been described in the literature. ADP-formingacetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples theconversion of acyl-CoA esters to their corresponding acids with theconcomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encodedby AF1211, was shown to operate on a variety of linear andbranched-chain substrates including isobutyrate, isopentanoate, andfumarate (Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). Asecond reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, wasalso shown to have a broad substrate range with high activity on cycliccompounds phenylacetate and indoleacetate (Musfeldt and Schonheit, J.Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui(annotated as a succinyl-CoA synthetase) accepts propionate, butyrate,and branched-chain acids (isovalerate and isobutyrate) as substrates,and was shown to operate in the forward and reverse directions (Brasenand Schonheit, Arch. Microbiol 182:277-287 (2004)). The ACD encoded byPAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilumshowed the broadest substrate range of all characterized ACDs, reactingwith acetyl-CoA, isobutyryl-CoA (preferred substrate) andphenylacetyl-CoA (Brasen and Schonheit, Arch. Microbiol 182:277-287(2004)). Directed evolution or engineering can be used to modify thisenzyme to operate at the physiological temperature of the host organism.The enzymes from A. fulgidus, H. marismortui and P. aerophilum have allbeen cloned, functionally expressed, and characterized in E. coli(Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004); and Musfeldtand Schonheit, J. Bacteriol. 184:636-644 (2002)). An additionalcandidate is the enzyme encoded by sucCD in E. coli, which naturallycatalyzes the formation of succinyl-CoA from succinate with theconcomitant consumption of one ATP, a reaction which is reversible invivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

GenBank Gene name GI# Accession # Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 AF1983 11499565 NP_070807.1Archaeoglobus fulgidus DSM 4304 scs 55377722 YP_135572.1 Haloarculamarismortui ATCC 43049 PAE3250 18313937 NP_560604.1 Pyrobaculumaerophilum str. IM2 sucC 16128703 NP_415256.1 Escherichia coli sucD1786949 AAC73823.1 Escherichia coli

Another candidate enzyme for this step is 6-carboxyhexanoate-CoA ligase,also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturallyactivates pimelate to pimeloyl-CoA during biotin biosynthesis ingram-positive bacteria. The enzyme from Pseudomonas mendocina, clonedinto E. coli, was shown to accept the alternate substrates hexanedioateand nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)).Other candidates are found in Bacillus subtilis (Bower et al., J.Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerlyBacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690(1992)).

GenBank Gene name GI# Accession # Organism pauA 15596214 NP_249708.1Pseudomonas mendocina bioW 50812281 NP_390902.2 Bacillus subtilis bioW115012 P22822.1 Lysinibacillus sphaericus

Additional CoA-ligases include the rat dicarboxylate-CoA ligase forwhich the sequence is yet uncharacterized (Vamecq et al., Biochem. J230:683-693 (1985)), either of the two characterized phenylacetate-CoAligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J395:147-155 (2006); and Wang et al., Biochem. Biophys. Res. Commun.360:453-458 (2007)) and the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J Biol. Chem. 265:7084-7090 (1990)).Additional candidate enzymes are acetoacetyl-CoA synthetases from Musmusculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008))and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003))which naturally catalyze the ATP-dependant conversion of acetoacetateinto acetoacetyl-CoA.

GenBank Gene name GI# Accession # Organism phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida AACS 21313520NP_084486.1 Mus musculus AACS 31982927 NP_076417.2 Homo sapiens

Example XXII Demonstration of Escherichia coli Tolerance to6-Aminocaproate

E. coli was assayed for tolerance, metabolic activity and growth duringexposure to various concentrations of 6-aminocaproate (6-ACA).Aerobically, cultures were able to grow media with up to 10% 6-ACA,while anaerobic cultures could grow in media with approximately 6% 6-ACA(FIG. 15). Because the pathway for producing 6-ACA could requireanaerobic conditions, all other further testing was performed underanaerobic conditions. To assay tolerance, cultures were grownanaerobically to mid-log (0.3 OD) and early stationary phase (0.6 OD),the cells were spun down and resuspended in medium containing variousconcentrations of 6-ACA. The cultures were grown in capped microfugetubes, grown overnight and the ODs of the cultures were assayed (FIG.16). Under these conditions, cultures were able to grow (double at least1 time) in up to 10% 6-ACA. The additional tolerance could have beenfrom the additional glucose from resuspending the cultures in freshM9-glucose medium or from limited oxygen that was present in the cappedmicrofuge tube. To determine if the cells were metabolically active inthe presence of 6-ACA, samples were taken and assayed for ethanolproduction (FIG. 17). Ethanol production (and thus metabolic activity)closely tracked with OD suggesting that if cells are present, they arelikely to be metabolically active. This is helpful to understand becauseit suggests that even though cells may be growth inhibited by theaccumulation of a product, they can still continue to produce product.

At high concentrations (>65 g/L) the osmolarity of 6-ACA is ˜0.5 M whichmay cause osmotic stress. To determine osmotic stress as the basis for6-ACA growth inhibition, cultures were grown in various concentrationsof 6-ACA with and without the osmoprotectant glycine betaine. As seen inFIG. 18, anaerobic growth in medium with up to 10-12% 6-ACA can beachieved if glycine betaine is present but only 4-6% without glycinebetaine. Therefore much of the toxicity of 6-ACA is likely due to theosmotic stress. However, it should be noted that 6-ACA is similar to theamino acid lysine and could have a greater toxic effect in the cellcytoplasm vs. outside the cell.

Example XXIII Demonstration of Enzyme Activity for CondensingSuccinyl-CoA and Acetyl-CoA to Form β-ketoadipyl-CoA

Several β-ketothiolase enzymes have been shown to break β-ketoadipyl-CoAinto acetyl-CoA and succinyl-CoA. For example, the gene products encodedby pcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol,184(1): 207-15 (2002)), phaD in Pseudomonas putida U (Olivera et al.,Proc Natl Acad Sci USA, 95(11), 6419-24 (1998)), paaE in Pseudomonasfluorescens ST (Di Gennaro et al., Arch Microbiol, 188(2), 117-25(2007)), and paaJ from E. coli (Nogales et al., Microbiology, 153(Pt 2),357-65 (2007)) catalyze the conversion of 3-oxoadipyl-CoA intosuccinyl-CoA and acetyl-CoA during the degradation of aromatic compoundssuch as phenylacetate or styrene. To confirm that β-ketothiolase enzymesexhibit condensation activity, several thiolases (Table 10; SEQ ID NOS:3-50, respectively) were cloned into a derivative of pZE13(Lutz et al.,Nucleic Acids Res, 29(18), 3873-81 (2001)), which results in the cloneshaving a carboxy-terminal 6×His tag (SEQ ID NO: 2).

TABLE 10 Cloned Thiolases Species Enzyme template Gene Length 5′ PRIMER3′ PRIMER ORF SEQ beta- Ralstonia bktB 1185 ATGACGCGTG GATACGCTCGatgacgcgtgaagtggtagtggtaagcggtgtccgta ketothiolase eutropha  AAGTGGTAGTAAGATGGCGG ccgcgatcgggacctttggcggcagcctgaaggatgt H16 GGTAAG (SEQ IDggcaccggcggagctgggcgcactggtggtgcgcgag (SEQ ID NO: 4)gcgctggcgcgcgcgcaggtgtcgggcgacgatgtcg NO: 3)gccacgtggtattcggcaacgtgatccagaccgagccgcgcgacatgtatctgggccgcgtcgcggccgtcaacggcggggtgacgatcaacgcccccgcgctgaccgtgaaccgcctgtgcggctcgggcctgcaggccattgtcagcgccgcgcagaccatcctgctgggcgataccgacgtcgccatcggcggcggcgcggaaagcatgagccgcgcaccgtacctggcgccggcagcgcgctggggcgcacgcatgggcgacgccggcctggtcgacatgatgctgggtgcgctgcacgatcccttccatcgcatccacatgggcgtgaccgccgagaatgtcgccaaggaatacgacatctcgcgcgcgcagcaggacgaggccgcgctggaatcgcaccgccgcgcttcggcagcgatcaaggccggctacttcaaggaccagatcgtcccggtggtgagcaagggccgcaagggcgacgtgaccttcgacaccgacgagcacgtgcgccatgacgccaccatcgacgacatgaccaagctcaggccggtcttcgtcaaggaaaacggcacggtcacggccggcaatgcctcgggcctgaacgacgccgccgccgcggtggtgatgatggagcgcgccgaagccgagcgccgcggcctgaagccgctggcccgcctggtgtcgtacggccatgccggcgtggacccgaaggccatgggcatcggcccggtgccggcgacgaagatcgcgctggagcgcgccggcctgcaggtgtcggacctggacgtgatcgaagccaacgaagcctttgccgcacaggcgtgcgccgtgaccaaggcgctcggtctggacccggccaaggttaacccgaacggctcgggcatctcgctgggccacccgatcggcgccaccggtgccctgatcacggtgaaggcgctgcatgagctgaaccgcgtgcagggccgctacgcgctggtgacgatgtgcatcggcggcgggcagggcattgccgccatcttcgagcgtatct ga (SEQ ID NO: 5) 2- Mus  ACA 1215ATGGAAGTAA CAGCTTCTCA atggaagtaagatgcctggaacgaagttatgcatcc Methyl-musculus T1 GATGCCTGGA ATCAGCAGGG aaacccactttgaatgaagtggttatagtaagtgctacetoacetyl ACGAAG C ataagaactcccattggatccttcctgggcagcctt CoA Thiolase(SEQ ID (SEQ ID gcctctcagccggccactaaacttggtactgctgca (branched NO: 6)NO: 7) attcagggagccattgagaaggcagggattccaaaa chain?)gaagaagtgaaggaagtctacatgggcaatgtcatccaagggggtgaaggacaggcccctaccaggcaagcaacactgggcgcaggtttacctatttccactccatgcaccacagtaaacaaggtttgtgcttcaggaatgaaagccatcatgatggcctctcaaagtcttatgtgtggacatcaggatgtgatggtggcaggcgggatggagagcatgtccaatgtcccatacgtaatgagcagaggagcaacaccatatggtggggtaaaacttgaagacctgattgtaaaagacgggctaactgatgtctacaataaaattcatatgggtaactgtgctgagaatactgcaaagaagatgaatatctcacggcaggaacaggatacgtacgctctcagctcttacaccagaagtaaagaagcgtgggacgcagggaagtttgccagtgagattactcccatcaccatctcagtgaaaggtaaaccagatgtggtggtgaaagaagatgaagaatacaagcgtgttgactttagtaaagtgccaaagctcaagaccgtgttccagaaagaaaatggcacaataacagctgccaatgccagcacactgaacgatggagcagctgctctggttctcatgactgcagaggcagcccagaggctcaatgttaagccattggcacgaattgcagcatttgctgatgctgccgtagaccccattgattttccacttgcgcctgcatatgccgtacctaaggttcttaaatatgcaggactgaaaaaagaagacattgccatgtgggaagtaaatgaagcattcagtgtggttgtgctagccaacattaaaatgctggagattgacccccaaaaagtaaatatccacggaggagctgtttctctgggccatccaattgggatgtctggagcccggattgttgttcatatggctcatgccctgaagccaggagagttcggtctggctagtatttgcaacggaggaggaggtgct tccgccctgctgattgagaagctgtag(SEQ ID NO: 8) 2- Pseudomonas fadAx 1194 ATGACCCTCG GTACAGGCATatgaccctcgccaatgaccccatcgttatcgtcagc Methyl- putida  CCAATGACCCTCAACAGCCA gccgtgcgcacgcccatgggcgggttgcagggcgac acetoacetyl- (KT2440)(SEQ ID TGG ctcaagagcctgactgcgccgcaactgggcagcgcc CoA Thiolase NO: 9)(SEQ ID gccattcgtgctgccgtggaacgggccggcatcgat (branched NO: 10)gccgccggtgtcgagcaggtactgttcggctgcgtg chain?)ctgccggccggccagggccaggcaccggcacgccaggccgcgctgggcgccgggctggacaagcacaccacctgcaccaccctgaacaagatgtgcggctcgggtatgcaagccgcgatcatggcccatgacctgctgctggccggcaccgcagacgtggtagtggcgggtggcatggaaagcatgaccaacgcgccgtacctgctggacaaagcccgtggcggctaccgcatgggccacggcaagatcatcgaccacatgttcatggacggtctcgaagacgcctacgacaaaggccgcctgatgggtacctttgccgaggactgtgcccaggccaatgccttcagccgcgaggcccaggaccagttcgccatcgcctcgctgacccgagcgcgagaagccatcagcagcggccgttttgccgccgagatcgtgccggtggaagtcaccgagggcaaggaaaagcgcgtcatcaaggatgacgagcagccgcccaaggcgcgtctggacaagattgcgcagctcaaaccggcgtttcgtgaaggcggcaccgtgacggcggccaacgccagttcgatttccgacggcgctgcggcgctggtactgatgcgccgctccgaggccgacaaacgtggcctcaagccattggccgtcatccacggccacgccgcctttgccgacaccccggcgctgttcccgaccgccccgatcggcgcgatcgacaaactgatgaaacgcaccggctggaacctggccgaagtcgacctgttcgagatcaacgaggccttcgccgtggtcaccctggcggccatgaaacacctcgacctgccacacgacaaggtcaatatccacggcggcgcctgcgccctcggtcacccgatcggcgcttctggcgcacgtattctggtcaccctgttgtcggccttgcgccagaacaatctgcgtcggggtgtggcggccatctgcatcggcggtggcgaggccacggccatggctgttgaatgcctg tactga (SEQ ID NO: 11) beta-Caenor- kat-1 1167 ATGAACAAAC TAATTTCTGGatgaacaaacatgctttcatcgtcggagccgcccgt ketothiolase habditis ATGCTTTCATCATAACCATTC acacctattggatcatttcgttcttctctctcttcg elegans GTCG CACTTGAGCgtaactgctccagagctcgcctcggttgccatcaaa (SEQ ID (SEQ IDgcagcattggagcgtggagcagtgaagccgagttca NO: 12) NO: 13)attcaggaggtgttccttggtcaagtctgtcaagcaaatgctggtcaagctcccgctcgtcaagcagctcttggagccggactcgatctttcggttgctgttaccaccgtcaataaagtgtgctcttctgggctgaaagcaatcattcttgctgcccagcaaattcaaaccggtcatcaagattttgccattggcggaggaatggagagcatgtcacaagtaccattttatgttcaaagaggagagatcccatatggtggatttcaagtgattgatggaatcgtcaaagacggactgaccgatgcttatgataaagttcacatgggaaactgcggagagaagacttcaaaagaaatgggaattacacgtaaagaccaagacgaatatgctatcaacagctacaaaaagtcagctaaagcatgggagaatggaaatatcggaccagaagtggtgccagtgaacgtcaaatcaaagaagggagtcacgattgttgataaagatgaagagttcacaaaagtcaatttcgacaagttcacctcgctgagaactgttttccagaaagacggaactatcactgctgctaatgcttcaacattgaacgacggtgcagctgctgtcattgttgcctcacaggaagcagtttccgagcaaagcttaaagcctctggcccgaattttggcttatggagatgccgccacgcacccactcgatttcgctgtagcaccaactttgatgttcccaaaaattcttgaaagagcaggagtgaagcaatcagatgttgctcaatgggaagttaatgaagccttctcatgtgttccccttgctttcatcaaaaaactaggagtcgatccatcccttgtgaacccacatggaggagctgtttcaattggtcaccccatcggaatgtccggagcccgcctcatcactcatcttgtgcacacactcaaaagtggccaaatcggagttgctgccatttgcaatggaggtggtggctcaagtggaatggtt atccagaaattataa (SEQ ID NO: 14)beta- Escherichia paaJ 1206 ATGCGTGAAG AACACGCTCCatgcgtgaagcctttatttgtgacggaattcgtacg ketothiolase coli CCTTTATTTGTAGAATCATGG ccaattggtcgctacggcggggcattatcaagtgtt NP_415915.1 GACG CGcgggctgatgatctggctgctatccctttgcgggaa (SEQ ID (SEQ IDctgctggtgcgaaacccgcgtctcgatgcggagtgt NO: 15) NO: 16)atcgatgatgtgatcctcggctgtgctaatcaggcgggagaagataaccgtaacgtagcccggatggcgactttactggcggggctgccgcagagtgtttccggcacaaccattaaccgcttgtgtggttccgggctggacgcactggggtttgccgcacgggcgattaaagcgggcgatggcgatttgctgatcgccggtggcgtggagtcaatgtcacgggcaccgtttgttatgggcaaggcagccagtgcattttctcgtcaggctgagatgttcgataccactattggctggcgatttgtgaacccgctcatggctcagcaatttggaactgacagcatgccggaaacggcagagaatgtagctgaactgttaaaaatctcacgagaagatcaagatagttttgcgctacgcagtcagcaacgtacggcaaaagcgcaatcctcaggcattctggctgaggagattgttccggttgtgttgaaaaacaagaaaggtgttgtaacagaaatacaacatgatgagcatctgcgcccggaaacgacgctggaacagttacgtgggttaaaagcaccatttcgtgccaatggggtgattaccgcaggcaatgcttccggggtgaatgacggagccgctgcgttgattattgccagtgaacagatggcagcagcgcaaggactgacaccgcgggcgcgtatcgtagccatggcaaccgccggggtggaaccgcgcctgatggggcttggtccggtgcctgcaactcgccgggtgctggaacgcgcagggctgagtattcacgatatggacgtgattgaactgaacgaagcgttcgcggcccaggcgttgggtgtactacgcgaattggggctgcctgatgatgccccacatgttaaccccaacggaggcgctatcgccttaggccatccgttgggaatgagtggtgcccgcctggcactggctgccagccatgagctgcatcggcgtaacggtcgttacgcattgtgcaccatgtgcatcggtgtcggtcagggcatcgccatg attctggagcgtgtttga (SEQ ID NO: 17)beta- Pseudomonas phaD 1221 ATGAATGAAC GAGGCGCTCGatgaatgaaccgacccacgccgatgccttgatcatc ketothiolase putida  CGACCCACGCATGATCATGG gacgccgtgcgcacgcccattggccgctatgccggg AAN68887.1 (KT2440) C(SEQ ID gccctgagcagcgtgcgcgccgacgacctggcggcc (SEQ ID NO: 19)atcccgctcaaagccttgatccagcgtcaccccgaa NO: 18)ctggactggaaagccattgatgacgttatcttcggctgtgccaaccaggctggcgaagacaaccgcaacgtggcccacatggcgagcctgctggccgggctgccactcgaagtaccagggaccacgatcaaccgcctgtgcggttccggtctggatgccatcggtaatgcggcacgtgccctgcgctgcggtgaagcggggctcatgctggccggtggtgtggagtccatgtcgcgtgcaccgtttgtgatgggtaagtcggagcaggcattcgggcgtgcggccgagctgttcgacaccaccatcggctggcgtttcgtcaacccgctgatgaaggccgcctacggcatcgattcgatgccggaaacggctgaaaacgtggccgaacagttcggcatctcgcgcgccgaccaggatgcctttgccctgcgcagccagcacaaagccgcagcagctcaggcccgcggccgcctggcgcgggaaatcgtgccggtcgaaatcccgcaacgcaaaggcccagccaaagtggtcgagcatgacgagcacccgcgcggcgacacgaccctggagcagctggctcggctcgggacgccgtttcgtgaaggcggcagcgtaacggcgggtaatgcctccggcgtgaatgacggcgcttgcgccctgctgctggccagcagcgccgcggcccgccgccatgggttgaaggcccgcggccgcatcgtcggcatggcggtggccggggttgagcccaggctgatgggcattggtccggtgcctgcgacccgcaaggtgctggcgctcaccggcctggcactggctgacctggatgtcatcgaactcaatgaggcctttgccgcccaagggctggccgtgttgcgcgagctgggcctggccgacgacgacccgcgagtcaaccgcaacggcggcgccatcgccctgggccatcccctgggcatgagcggtgcccggttggtgaccactgccttgcacgagcttgaagaaacggccggccgctacgccctgtgcaccatgtgcatcggcgtaggc caaggcattgccatgatcatcgagcgcctctga(SEQ ID NO: 20) beta- Clostridium thiA 1179 ATGAAAGAAG GCACTTTTCTatgaaagaagttgtaatagctagtgcagtaagaaca ketothiolase aceto- TTGTAATAGCTAGCAATATTG gcgattggatcttatggaaagtctcttaaggatgta NP_349476.1 butylicumAGTGCAGTAA CTGTTCC  ccagcagtagatttaggagctacagctataaaggaa ATCC 824 GAAC(SEQ ID gcagttaaaaaagcaggaataaaaccagaggatgtt (SEQ ID NO: 22)aatgaagtcattttaggaaatgttcttcaagcaggt NO: 21)ttaggacagaatccagcaagacaggcatcttttaaagcaggattaccagttgaaattccagctatgactattaataaggtttgtggttcaggacttagaacagttagcttagcagcacaaattataaaagcaggagatgctgacgtaataatagcaggtggtatggaaaatatgtctagagctccttacttagcgaataacgctagatggggatatagaatgggaaacgctaaatttgttgatgaaatgatcactgacggattgtgggatgcatttaatgattaccacatgggaataacagcagaaaacatagctgagagatggaacatttcaagagaagaacaagatgagtttgctcttgcatcacaaaaaaaagctgaagaagctataaaatcaggtcaatttaaagatgaaatagttcctgtagtaattaaaggcagaaagggagaaactgtagttgatacagatgagcaccctagatttggatcaactatagaaggacttgcaaaattaaaacctgccttcaaaaaagatggaacagttacagctggtaatgcatcaggattaaatgactgtgcagcagtacttgtaatcatgagtgcagaaaaagctaaagagcttggagtaaaaccacttgctaagatagtttcttatggttcagcaggagttgacccagcaataatgggatatggacctttctatgcaacaaaagcagctattgaaaaagcaggttggacagttgatgaattagatttaatagaatcaaatgaagcttttgcagctcaaagtttagcagtagcaaaagatttaaaatttgatatgaataaagtaaatgtaaatggaggagctattgcccttggtcatccaattggagcatcaggtgcaagaatactcgttactcttgtacacgcaatgcaaaaaagagatgcaaaaaaaggcttagcaactttatgtataggtggcggacaagga acagcaatattgctagaaaagtgctag(SEQ ID NO: 23) beta- Clostridium thiB 1179 ATGAGAGATG GTCTCTTTCAatgagagatgtagtaatagtaagtgctgtaagaact ketothiolase aceto- TAGTAATAGTACTACGAGAG gcaataggagcatatggaaaaacattaaaggatgta NP_149242.1 butylicumAAGTGCTGTA CTGTTCCC cctgcaacagagttaggagctatagtaataaaggaa ATCC 824AGAACTG (SEQ ID gctgtaagaagagctaatataaatccaaatgagatt (SEQ ID NO: 25)aatgaagttatttttggaaatgtacttcaagctgga NO: 24)ttaggccaaaacccagcaagacaagcagcagtaaaagcaggattacctttagaaacacctgcgtttacaatcaataaggtttgtggttcaggtttaagatctataagtttagcagctcaaattataaaagctggagatgctgataccattgtagtaggtggtatggaaaatatgtctagatcaccatatttgattaacaatcagagatggggtcaaagaatgggagatagtgaattagttgatgaaatgataaaggatggtttgtgggatgcatttaatggatatcatatgggagtaactgcagaaaatattgcagaacaatggaatataacaagagaagagcaagatgaattttcacttatgtcacaacaaaaagctgaaaaagccattaaaaatggagaatttaaggatgaaatagttcctgtattaataaagactaaaaaaggtgaaatagtctttgatcaagatgaatttcctagattcggaaacactattgaagcattaagaaaacttaaacctattttcaaggaaaatggtactgttacagcaggtaatgcatccggattaaatgatggagctgcagcactagtaataatgagcgctgataaagctaacgctctcggaataaaaccacttgctaagattacttcttacggatcatatggggtagatccatcaataatgggatatggagctttttatgcaactaaagctgccttagataaaattaatttaaaacctgaagacttagatttaattgaagctaacgaggcatatgcttctcaaagtatagcagtaactagagatttaaatttagatatgagtaaagttaatgttaatggtggagctatagcacttggacatccaataggtgcatctggtgcacgtattttagtaacattactatacgctatgcaaaaaagagattcaaaaaaa ggtcttgctactctatgtattggtggaggtcagggaacagctctcgtagttgaaagagactaa (SEQ ID NO: 26) 3-oxoadipyl- Candida POT981182 ATGTTCAAGA CTCGTTAGCA atgttcaagaaatcagctaatgatattgttgttatCoA thiolase albicans AATCAGCTAA AACAAGGCAGtgcagcaaagagaactccaatcaccaagtcaatta SC5314 TGATATTGTTG CGaaggtgggttgagtagattatttcctgaggaaata (SEQ ID (SEQ IDttatatcaagtggttaagggtactgtatcagattc NO: 27) NO: 28)acaagttgatttaaacttgattgatgatgtgttag tcggtacggtcttgcaaactttagggggacagaaagctagtgccttggccattaaaaagattggattccc aattaagaccacggttaatacggtcaatcgtcaatgtgctagttctgctcaagcgattacttatcaagca ggtagtttgcgtagtggggagaatcaatttgctattgctgctggagtagaaagtatgactcatgattatt ttcctcatcgtgggattcccacaagaatttctgaatcatttttagctgatgcatccgatgaagctaaaaa cgtcttgatgccaatggggataaccagtgaaaatgttgccactaaatatggaatttctcgtaaacaacaa gatgagtttgcccttaattctcatttgaaagcagacaaggctacaaaactgggtcattttgcaaaagaaa tcattcctattcaaacaacggatgaaaacaaccaacacgtttcaataaccaaagatgatggtataagggg aagttcaacaattgaaaagttgggtggcttaaaacctgtgttcaaggatgatgggactactactgctggt aattcctcgcaaatttcagatggagggtctgctgtgattttaactactcgtcaaaatgctgagaaatcgg gagtaaagccaatagctagatttattggttcgtcagtagctggtgttccttcgggacttatgggaattgg tccatcggctgctattcctcaattgttgtcgagattaaatgttgacacgaaagacattgatatttttgaa ttgaacgaggcatttgcatcccaactgatttattgtattgaaaaattgggtcttgattatgataaagtca atccatatggtggagctatagccttgggacatccattaggagccactggcgcaagagttacggcaacgtt gcttaatggattaaaagatcagaataaagagttgggtgtcatctcaatgtgcacatccacaggtcaagga tacgctgccttgtttgctaacgagtag(SEQ ID NO: 29) 3-oxoadipyl- Candida POT1 1227 ATGGATAGAT TTCCTTAATCatggatagattaaatcaattaagtggtcaattaaa CoA thiolase albicans TAAATCAATTAATATGGAGG accaacttcaaaacaatcccttactcaaaagaacc SC5314 AAGTGGTCAA CAGCACcagacgatgttgtcatcgttgcagcatacagaact TTAAAACC (SEQ IDgccatcggtaaaggtttcaaagggtctttcaaatc (SEQ ID NO: 31)tgtgcaatctgaattcatcttgactgaattcttga NO: 30)aagaatttattaaaaagactggagtcgatgcatct ttgattgaagatgttgctattggtaacgttttgaaccaagctgctggtgccaccgaacacagaggtgcta gtttggctgcaggtattccttacactgcagctttccttgccatcaacagattgtgttcctcagggttaat ggccatttctgacattgccaacaaaatcaaaaccggtgaaatcgaatgtggtcttgctggtggtattgaa tccatgtctaaaaactatggtagtccaaaagttattccaaagattgacccacacttggctgatgacgaac aaatgagtaaatgtttgattccaatgggtatcaccaacgaaaatgttgctaatgaattcaacattccaag agaaaaacaagatgcctttgctgctaaatcttatagtaaagccgaaaaagccatctcctctggagctttc aaagatgaaatcttaccaatcagatccattatcagatccccagacggttctgaaaaagaaatcattgtcg ataccgacgaaggtccaagaaagggtgttgacgctgcttccttgagcaaattgaaaccagcatttggtgg tactaccactgccggtaacgcttctcaaatttcagatggtgctgctggtgttttattgatgaagagaagt ttggctgaagccaaaggttacccaattgttgctaaatacattgcttgttcaactgttggtgttccgccag aaatcatgggtgttggtccagcttacgccattccagaagtgttgaagagaactggattgactgtggatga cgttgatgtgtttgaaatcaacgaagcttttgctgctcaatgtctttactcagctgaacaatgtaatgtt ccagaagaaaaattgaacataaacggtggtgccatcgctttaggtcatcctcttggttgtactggtgcca gacaatatgccactatcttgagattgttgaaaccaggtgaaattggtttgacttctatgtgtatcggtag tggtatgggtgctgcctccatattgattaaggaatag (SEQ ID NO: 32) 3-oxoadipyl- Candida POT2 1233 ATGTCATCCA TTCTCTAACCatgtcatccaaacaacaatacttgaagaagaatc CoA thiolase albicans AACAACAATAAAAACAGAAG ctgacgatgtcgttgtcgttgcagcatacagaac SC5314 CTTGAAGAAG CAGCACCtgctttaaccaaaggtggaagaggtggattcaaa (SEQ ID (SEQ IDgatgttggatctgatttccttttgaaaaaattga NO: 33) NO: 34)ctgaagaatttgttaaaaaaactggtgttgaccc taaaatcattcaagatgctgccattggtaatgtcttgaacagaagagctggtgatttcgaacatagag gtgcattattatctgctggattaccttattcagttccatttgttgcccttaacagacaatgttcatct gggttaatggccatttctcaagtggccaacaagatcaagactggtgaaattgaatgtggtttagctgg tggtgttgaaagtatgacaaaaaactatggtccagaagcattgattgctattgaccctgcttatgaaa aagacccagaatttgttaaaaacggtattccaatgggtattactaatgaaaatgtttgtgccaaattc aatatttcaagagatgttcaagatcaatttgctgctgaatcttatcaaaaagctgaaaaggcacaaaa agaaggtaaatttgatgatgaaattttaccaattgaagttttccaagaagatgaagatgctgaagatg aagacgaagatgaagatgaagatgctgaaccaaaagaaaaattggttgttattagtaaagatgaaggt attagaccaggtgttactaaagaaaaattggctaaaattaaaccagctttcaaatctgatggtgtatc ttcagctggtaactcttcacaagtttccgatggtgctgccttggtgttattgatgaaacgttcatttg ctgaaaagaatggattcaaaccattggctaaatacatttcttgtggtgttgctggtgtcccaccagaa attatgggtattggtccagctgttgccattccaaaagttttgaaacaaactggattatcagtcagtga tattgatatttatgaaatcaatgaagcatttgccggtcaatgtttgtactcaattgaaagttgtaata ttccaagagaaaaagtcaatcttaatgggggtgctattgccttgggtcaccctcttggttgtactggt gctagacaatacgctactattttaagattgttaaaaccaggtgaatttggtgtgacttctatgtgtat tggtactggtatgggtgctgcttctgttttggttagagaataa (SEQ ID NO: 35) beta- Pseudomonas pcaF 1206 ATGAGCCGCGGACCCGCTCG atgagccgcgaggtattcatctgcgatgccgtgc ketoadipyl aeruginosa AGGTATTCAT ATGGCCAG gcacgccgatcggccgtttcggcggcagtctttc CoA thiolase PAO1CTG (SEQ ID cgcggtgcgcgccgacgacctcgcggcggtgccg pcaF (SEQ ID NO: 37)ctgaaggccctggtcgagcgcaacccgggggtcg NO: 36)actggtcggcgttggacgaggtgttcctcggctg cgccaaccaggccggcgaggacaaccgtaacgtggcgcgcatggcgctgctgctggccggtttgccgg agagcgtgcccggcgtcaccctcaaccgcctctgcgcctcggggatggacgccatcggcacggcgttc cgcgccatcgcctgcggcgagatggagctggccatcgccggcggcgtcgagtcgatgtcgcgcgcgcc gtacgtgatgggcaaggccgatagcgccttcggtcgcggccagaagatcgaggacaccaccatcggct ggcgcttcgtcaatccgctgatgaaggagcagtacggcatcgacccgatgccgcagaccgccgacaac gtcgccgacgactatcgcgtgtcgcgtgccgaccaggatgccttcgccctgcgcagccagcagcgcgc cggcagggcgcaggaggccggtttcttcgccgaggaaatcgtcccggtgacgattcgcgggcgcaagg gcgacaccctggtcgagcacgacgagcatccgcgtcccgacaccaccctggaggcgctggcccggctc aagccggtcaacgggccggagaagaccgtcaccgccggcaacgcgtccggggtcaacgacggcgccgc cgcgctggtcctggcctccgccgaggcagtggagaagcacggcctgactccgcgcgcgcgggtgctgg gcatggccagcgccggcgtcgccccacggatcatgggcatcggcccggtgccggcggtgcgcaagctg ctgcggcgcctggacctggcgatcgacgccttcgacgtgatcgaactcaacgaagccttcgccagcca gggcctggcctgcctgcgcgaactgggcgtggccgacgacagtgagaaggtcaacccgaacggcggtg ccatcgccctcggccacccgctggggatgagcggtgcgcggctggtcctcaccgcgctccatcaactt gagaagagcggcggccggcgcggcctggcgaccatgtgcgtaggcgtcggccaaggcctggcgctggc catcgagcgggtctga (SEQ ID NO: 38)acyl-CoA Pseudomonas bkt 1206 ATGCTCGATG TCGGCAGCGCatgctcgatgcctatatctacgccggcctgcgta thiolase aeruginosa  CCTATATCTACTCGATCAC cgcctttcggccggcatgccggtgcactctcgac PAO1 GCC (SEQ IDggtgcgtccggacgacctggccggcctgctgctg (SEQ ID NO: 40)gcgcgtctcgcggaaacctccgggttcgccgtcg NO: 39)acgacctggaggatgtgatcctcggttgcaccaa ccaggccggcgaagacagccgcaacctggcgcgcaacgcgctgctcgcagccggcctgccggcgcggc tgcccgggcagacggtcaaccgcttgtgtgccagcggactgtcggcggtgatcgacgcggcgcgcgcg atcagttgcggtgagggccggctgtacctggccggcggcgccgaaagcatgtcccgggcgccgttcgt catgggcaaggcggagagcgccttcagccgcacgctggaggtcttcgacagcaccatcggcgcgcgct tcgccaaccccaggctggtcgagcgctatggcaacgacagcatgccggagaccggcgacaacgtggcc cgcgccttcggcatcgcccgcgaagacgccgaccgtttcgccgcttcttcccaggcgcgctaccaggc tgcgctggaggagggctttttcctcggcgagatccttccggtggaggtgcgtgccggacgcaagggcg agacgcggctggtggagcgcgacgagcatccgcgaccgcaggccgacctggcggccctggcgcgcttg ccggcgttgttcgccggtggggtagtgaccgccggtaatgcgtctgggatcaacgacggggcggcggt agtgctgctgggcgatcgcgcgatcggcgagcgcgagggcatccggccgttggcgcggatcctcgcca gcgccagcgtcggcgtcgagccccggttgatgggcatcggcccgcagcaggcgatcctccgcgcgctg caacgcgccggcatcgacctggacgaggtcggcctgatcgagatcaacgaagccttcgcgccgcaggt cctggcctgcctgaagttgctcggcctggactacgaggacccgcgggtcaatccccatggcggcgcca ttgccctcggccatccgctcggcgcctccggtgcgcgcctggtgctcaccgccgcccgcgggctgcaa cgcatcgagcggcgctacgcggtggtcagcctgtgcgtcgggctcggccagggcgtggcgatggtgat cgagcgctgccgatga (SEQ ID NO: 41)3-oxoadipyl- Pseudomonas pcaF 1203 ATGCACGACG AACCCGCTCGatgcacgacgtattcatctgtgacgccatccgta CoA thiolase putida  TATTCATCTGTATGGCCAAC ccccgatcggccgcttcggcggcgccctggccag (KT2440) GACG (SEQ ID cgtgcgggccgacgacctggccgccgtgccgctg (SEQ ID NO: 43)aaggcgctgatcgagcgcaaccctggcgtgcagt NO: 42)gggaccaggtagacgaagtgttcttcggctgcgc caaccaggccggtgaagacaaccgcaacgtggcccgcatggcactgctgctggccggcctgccggaaa gcatcccgggcgtcaccctgaaccgtctgtgcgcgtcgggcatggatgccgtcggcaccgcgttccgc gccatcgccagcggcgagatggagctggtgattgccggtggcgtcgagtcgatgtcgcgcgccccgtt cgtcatgggcaaggctgaaagcgcctattcgcgcaacatgaagctggaagacaccaccattggctggc gtttcatcaacccgctgatgaagagccagtacggtgtggattccatgccggaaaccgccgacaacgtg gccgacgactatcaggtttcgcgtgctgatcaggacgctttcgccctgcgcagccagcagaaggctgc cgctgcgcaggctgccggcttctttgccgaagaaatcgtgccggtgcgtatcgctcacaagaagggcg aaatcatcgtcgaacgtgacgaacacctgcgcccggaaaccacgctggaggcgctgaccaagctcaaa ccggtcaacggcccggacaagacggtcaccgccggcaacgcctcgggcgtgaacgacggtgctgcggc gatgatcctggcctcggccgcagcggtgaagaaacacggcctgactccgcgtgcccgcgttctgggca tggccagcggcggcgttgcgccacgtgtcatgggcattggcccggtgccggcggtgcgcaaactgacc gagcgtctggggatagcggtaagtgatttcgacgtgatcgagcttaacgaagcgttgccagccaaggc ctggcggtgctgcgtgagctgggtgtggctgacgatgcgccccaggtaaaccctaatggcggtgccat tgccctgggccaccccctgggcatgagcggtgcacgcctggtactgactgcgttgcaccagctggaga agagtggcggtcgcaagggcctggcgaccatgtgtgtgggtgtcggccaaggtctggcgttggccatc gagcgggtttga (SEQ ID NO: 44)3-oxoadipyl- Burkholderia bkt 1203 ATGACCGACG CACGCGTTCGatgaccgacgcctacatctgcgatgcgattcgca CoA thiolase ambifaria  CCTACATCTGCATCGCGATC cacccatcggccgctacggcggcgccctgaaaga AMMD G (SEQ IDcgttcgtgccgacgatctcggcgcggtgccgctc (SEQ ID NO: 46)aaggcgctgatcgaacgcaaccggaacgtcgact NO: 45)ggtcggcgatcgacgacgtgatctatggctgcgc gaaccaggccggcgaagacaaccgcaacgtcgcgcgcatgtccgcgctgctcgcgggcttgccgaccg ccgtgccgggcacgacgctgaaccggttatgcggctcgggcatggacgccgtcggcacggccgcgcgc gcgatcaaggcgggcgaggcacgcttgatgatcgcgggcggcgtcgaaagcatgacgcgcgcgccgtt cgtgatgggcaaggccgccagcgcattcgcgcgccaggctgcgattttcgacacgacgatcggctggc gtttcattaatccgctgatgaaacagcaatacggcgtcgattcgatgcccgagacggccgagaacgtc gcggtcgactacaacatcagccgcgccgaccaggatctattcgcgctgcgcagccagcagaaggccgc gcgtgcgcagcaggacggcacgctcgccgccgaaatcgtccccgtcacgattgcgcagaaaaaaggcg acgcgctcgtcgtatcgctcgacgagcatccgcgcgaaacatcgctcgaagcgctcgcgaagctgaag ggcgtcgtgcgtcccgacggctcggtcacggccggcaacgcgtcaggcgtcaacgacggcgcatgcgc actgctgctcgccaacgcggaagccgccgatcaatatgggctgcgccgccgcgcgcgtgtcgtcggca tggcgagcgccggcgtcgagccgcgcgtgatgggtatcggcccggcgccggccacgcagaaactgttg cgccagctcggcatgacgatcgaccagttcgacgtgatcgagctgaacgaagcgttcgcgtcgcaggg tctcgcggtgctgcgcatgctcggtgtcgccgacgacgatccgcgcgtgaaccccaacggcggtgcga tcgcgctcggccatccgctcggcgcatcgggtgcgcggctcgtgaccacggcgcttcaccaactcgag cgtacgggcggccgctttgcgctctgtacgatgtgcatcggcgtcggccagggcatcgcgatcgcgat cgaacgcgtgtaa (SEQ ID NO: 47) beta-Ascaris bkt 1242 ATGGCCACCT CAATTTCTCGgtgatggccacctcaagacttgtctgcagcaatt ketothiolase suum CAAGACTTGTATGACCATTC taacgaagcaatgctttacgatctcgtcacgtgc CTGC CACCtgctagccaatttaccgatgtggtattcgtgggt (SEQ ID (SEQ IDgccgcacgaacaccggtcggatcgtttcgctctt NO: 48) NO: 49)cgctttccactgttccagccactgtcctcggagc tgaggctattaagggtgcacttaaacatgccaatctaaaaccctcacaagtgcaagaggtgttctttg gctgtgtcgttccatccaactgtggacaagttcctgcccgtcaagcgacacttggagctggatgcgat ccttcgacaatcgttacaactctcaataaattgtgcgcctcgggaatgaagtcgattgcttgtgccgc ctcacttttgcaacttggtcttcaagaggttaccgttggtggcggtatggagagcatgagcttagtgc cgtactatcttgaacgtggtgaaactacttatggtggaatgaagctcatcgacggtatcccaagagat ggtccgactgatgcatatagtaatcaacttatgggtgcatgcgctgataatgtggctaaacgattcaa catcacccgtgaggaacaggataaattcgctattgaaagctataaacgatctgctgctgcatgggaga gtggagcatgcaaagctgaagtagttcctattgaagtgacaaagggcaagaaaacatacattgtcaac aaggatgaggaatacatcaaagtcaacttcgagaagcttcccaaactgaaacccgccttcttgaaaga cggaaccatcacggctggcaatgcttcaacactgaacgatggtgctgcggcagttgtgatgacgactg tcgaaggagcgaaaaaatacggtgtgaaaccattggcccgattgctctcatatggtgatgcggcaaca aatccagtcgattttgctattgcaccatcaatggttatcccaaaggtacttaaattggctaatctcga gatcaaggatattgatttgtgggaaatcaacgaggctttcgccgttgttccccttcattcaatgaaga cactcggtatcgatcactcgaaagtgaacattcatggtggtggcgtatctcttggacatcctattgga atgtctggagctcgaattatcgttcatctgattcatgcgttgaaacctggccagaaaggctgcgctgc aatctgcaatggtggcggtggcgctggtggaatggtcatcgagaaattgtaa (SEQ ID NO: 50)

The genes were expressed in E. coli and the proteins purified usingNi-NTA spin columns and quantified. To assay enzyme activity in vitro, a5× CoA:DTNB (Ellman's reagent or 5, 5′-dithiobis-(2-nitrobenzoic acid))mixture was prepared. The mixture consisted of 10 mM succinyl-CoA, 5 mMacetyl-CoA, 30 mM DTNB in 100 mM Tris buffer, pH 7.4. Five μI, of theCoA:DTNB mixture was added to 0.5 μM purified thiolase enzyme in 100 mMTris buffer, pH 7.8 in a final volume of 50 μL. The reaction wasincubated at 30° C. for 30 minutes, then quenched with 2.5 μl, 10%formic acid and samples frozen at −20° C. until ready for analysis byLC/MS. Because many thiolases can condense two acetyl-CoA molecules intoacetoaceytl-CoA, production of acetoacetyl-CoA was examined. FIG. 19shows that 3 thiolases demonstrated thiolase activity which resulted inacetoacetyl-CoA formation. These were fadAx from Pseudomonas putida,thiA from Clostridium acetobutylicum and thiB also from Clostridiumacetobutylicum. When enzyme assays were examined for condensation ofsuccinyl-CoA and acetyl-CoA into β-ketoadipyl-CoA, several candidatesdemonstrated the desired activity; paaJ from Escherichia coli (Nogaleset al., Microbiol. 153:357-365 (2007)), phaD from Pseudomonas putida(Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), bktfrom Burkholderia ambifaria AMMD, pcaF from Pseudomonas putida KT2440(Harwood et al., J. Bacteriol. 176:6479-6488 (1994)), and pcaF fromPseudomonas aeruginosa PAO1. There was excellent specificity between thethiolases. Those that generated significant amounts of β-ketoadipyl-CoAdid not produce significant amounts of acetoacetyl-CoA and likewisethose that made acetoacetyl-CoA did not make detectable amounts ofβ-ketoadipyl-CoA.

Example XXIV Pathways for Production of Hexamethylenediamine fromGlutamate, Glutaryl-CoA or Pyruvate and 4-Aminobutanal

This example describes exemplary pathways for production ofhexamethylenediamine (HMDA) from glutamate, glutaryl-CoA, pyruvate and4-aminobutanal, or 2-amino-7-oxosubarate through homolysine, theseven-carbon analog of lysine. Homolysine is an attractive precursor toHMDA. Although homolysine is a potentially valuable precursor, it is nota known metabolic intermediate of any organism. Homolysine can be formedbiocatalytically from the central metabolic precursors glutamate,glutaryl-CoA or pyruvate and 4-aminobutanal. Subsequent decarboxylationof homolysine by an enzyme analogous to lysine decarboxylase yieldsHMDA.

This example describes additional pathways that proceed from2-amino-7-oxosubarate, or pyruvate and 4-aminobutanal through theintermediate 6-aminohexanal. 6-Aminohexanal can readily be converted toHMDA by an aminotransferase or an aminating oxidoreductase.

The maximum theoretical yield of HMDA is 0.71 moles per mole glucoseutilized (0.46 g/g). The pathways disclosed in FIGS. 20-22 and 26achieve a maximum HMDA yield of 0.67 mol/mol (0.43 g/g).

C₆H₁₂O₆+1.41NH₄→0.71C₆H₁₈N₂+1.76CO₂+2.47H₂O

Novel pathways for producing hexamethylenediamine (HMDA) and relatedproducts are described herein. The candidate enzymes, and associatedrisks of implementation are discussed in Example XXVI below.

This invention is directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyze HMDAproduction. Successfully engineering these pathways entails identifyingan appropriate set of enzymes with sufficient activity and specificity,cloning their corresponding genes into a production host, optimizing theexpression of these genes in the production host, optimizingfermentation conditions, and assaying for product formation followingfermentation.

HMDA can be produced from glutamate via glutaryl-CoA in eight enzymaticsteps, shown in FIG. 20. In this route, glutamate is acylated toglutamyl-CoA by a CoA transferase or ligase (Step A of FIG. 20).Glutamyl-CoA and acetyl-CoA are joined by a beta-ketothiolase to formthe C7 compound 3-oxo-6-aminopimeloyl-CoA (Step B of FIG. 20). The 3-oxogroup of this product is then reduced and dehydrated, resulting in6-amino-7-carboxyhept-2-enoyl-CoA (Steps C and D of FIG. 20). Anenoyl-CoA reductase reduces the double bond, forming 6-aminopimeloyl-CoA(Step E of FIG. 20). 6-Aminopimeloyl-CoA is then converted to2-amino-7-oxoheptanoate by a CoA-dependent aldehyde dehydrogenase (StepF). Transamination of the aldehyde to an amine yields homolysine (Step Gof FIG. 20). Finally, HMDA is formed as the decarboxylation product ofhomolysine (Step H of FIG. 20). The maximum theoretical HMDA yield forthis pathway is 0.67 moles of HMDA per mole of glucose utilized. Yieldcalculations assume aerobic conditions and the utilization of a CoAtransferase in Step A.

HMDA can also be produced from glutaryl-CoA by several routes. Exemplaryroutes for HMDA production are shown in FIG. 21. Glutaryl-CoA is acommon metabolic intermediate in organisms that metabolize aromaticcompounds. In the disclosed pathways to HMDA, glutaryl-CoA is firstcondensed with acetyl-CoA by a beta-ketothiolase to form3-oxopimeloyl-CoA (Step A of FIG. 21). The CoA moiety of3-oxopimeloyl-CoA is removed by a CoA hydrolase, transferase and ligase(Step B of FIG. 21). Several alternate routes for converting3-oxopimelate to HMDA are outlined in FIG. 21 and described herein. Thefinal step of all routes to HMDA entails decarboxylation of homolysine(Step S of FIG. 21).

One route entails conversion of 3-oxopimelate to3-oxo-1-carboxyheptanal. This conversion can be catalyzed by an ATP- andNAD(P)H dependent enzyme with 3-oxopimelate reductase activity (Step Cof FIG. 21), or alternately can proceed through activated intermediates5-oxopimeloyl-CoA (Steps H, I of FIG. 21) or 5-oxopimeloyl-phosphonate(Steps F, G of FIG. 21). Once formed, 3-oxo-1-carboxyheptanal istransaminated at the 3-position (Step AB of FIG. 21) or 7-position (StepD of FIG. 21). Subsequent transamination of 3-oxo-7-aminoheptanoate(Step E of FIG. 21) or 3-amino-7-oxoheptanoate (Step Z of FIG. 21)yields 3,7-diaminoheptanoate. An enzyme with 3,7-diaminoheptanoate2,3-aminomutase activity then forms homolysine (Step R of FIG. 21),which is decarboxylated to HMDA (Step S of FIG. 21).

In an alternate route, 3-oxopimelate is transaminated to 3-aminopimelate(Step J of FIG. 21). 3-Aminopimelate is then converted to3-amino-7-oxoheptanoate directly (Step O of FIG. 21) or via a CoA (StepsK, L of FIG. 21) or phosphonic acid (Steps M, N of FIG. 21)intermediate. 3-Amino-7-oxoheptanoate is subsequently converted to2-amino-7-oxoheptanoate by a 2,3-aminomutase (Step P of FIG. 21).2-Amino-7-oxoheptanoate is converted to homolysine by anaminotransferase or aminating oxidoreductase. Alternately,3-amino-7-oxoheptanoate is first transaminated (Step Z of FIG. 21) andthen converted to homolysine by an aminomutase (Step R of FIG. 21).

3-Aminopimelate can be converted to 2-aminopimelate by a 2,3-aminomutaseenzyme (Step T of FIG. 21). An HMDA pathway involving this intermediaterequires reduction of the 7-carboxylic acid to an aldehyde. Thisreduction is catalyzed by a bifunctional reductase (Step W of FIG. 21)or by two enzymes that proceed through a CoA (Steps V, Y of FIG. 21) orphosphonic acid (Steps U, X of FIG. 21) intermediate. The product,2-amino-7-oxoheptanoate is converted to HMDA as described above.

Two routes for producing HMDA from pyruvate and 4-aminobutanal are shownin FIG. 22. The routes achieve a maximum yield of 0.67 moles of HMDA permole glucose utilized (0.43 g/g) under anaerobic and aerobic conditions.4-Aminobutanal is naturally derived from ornithine by decarboxylation toputrescine and subsequent transamination. 4-Aminobutanal can alsooriginate from 4-aminobutanoate. In one pathway, 4-aminobutanal andpyruvate are joined by aldol condensation to form2-oxo-4-hydroxy-7-aminoheptanoate (Step A of FIG. 22). The condensationproduct is subsequently dehydrated (Step B of FIG. 22) and reduced (StepC of FIG. 22). Transamination of 2-oxo-7-aminoheptanoate yieldshomolysine (Step D of FIG. 22). HMDA is the decarboxylation product ofhomolysine decarboxylase (Step E of FIG. 22). Alternately, pathwayintermediate 2-oxo-7-aminoheptanoate is decarboxylated to form6-aminohexanal (Step F of FIG. 22). 6-Aminohexanal is subsequentlyconverted to HMDA by an aminotransferase or aminating oxidoreductase(Step G of FIG. 22).

Several routes for producing HMDA from 2-amino-7-oxosubarate are shownin FIG. 26. 2-Amino-7-oxosubarate is not known to be a naturallyoccurring metabolite. An exemplary route for synthesizing2-amino-7-oxosubarate is shown in FIG. 27. The pathway originates withglutamate-5-semialdehyde, a metabolite naturally formed during ornithinebiosynthesis. 2-Amino-7-oxosubarate is then synthesized in threeenzymatic steps. In the first step, glutamate-5-semialdehyde iscondensed with pyruvate by an aldolase (FIG. 27, Step A). The product,2-amino-5-hydroxy-7-oxosubarate is subsequently dehydrated and theresulting alkene is reduced to form 2-amino-7-oxosubarate (FIG. 27,Steps B/C). In one proposed pathway to HMDA from 2-amino-7-oxosubarate,the 2-oxo acid is first decarboxylated to form 2-amino-7-oxoheptanoate(Step A of FIG. 26). This product is again decarboxylated, forming6-aminohexanal (Step B of FIG. 26). Finally, 6-aminohexanal is convertedto HMDA by an aminotransferase or aminating oxidoreductase (Step C ofFIG. 26).

Alternately, the intermediate 2-amino-7-oxoheptanoate is first convertedto homolysine by an aminotransferase or aminating oxidoreductase (Step Mof FIG. 26). Homolysine is decarboxylated to HMDA as describedpreviously (Step H of FIG. 26).

In yet another route, the 2-amino acid group of 2-amino-7-oxosubarate isdecarboxylated, yielding 2-oxo-7-aminoheptanoate (Step I of FIG. 26).This product can then be further decarboxylated to 6-aminohexanal (StepG of FIG. 26) or transaminated to homolysine (Step J of FIG. 26).Homolysine or 6-aminohexanal is then converted to HMDA as describedpreviously.

In yet another route, the 2-oxo group of 2-amino-7-oxosubarate isconverted to an amino group, forming 2,7-diaminosubarate (Step K of FIG.26). Two subsequent decarboxylations yield HMDA (Steps L, H of FIG. 26).

Described herein is the generation of a microbial organism that has beenengineered to produce HMDA from pyruvate and 4-aminobutanal, as shown inFIG. 22 (Steps A-E). This example also teaches a method for engineeringa strain that overproduces HMDA.

Escherichia coli is used as a target organism to engineer aHMDA-producing pathway as shown in FIG. 22. E. coli provides a good hostfor generating a non-naturally occurring microorganism capable ofproducing HMDA. E. coli is amenable to genetic manipulation and is knownto be capable of producing various products, like ethanol, acetic acid,formic acid, lactic acid, and succinic acid, effectively underanaerobic, microaerobic or aerobic conditions.

An E. coli strain is engineered to produce HMDA from 4-aminobutanal viathe route outlined in FIG. 22. For the first stage of pathwayconstruction, genes encoding enzymes to transform 4-aminobutanal andpyruvate to homolysine (FIG. 3, Steps A-D) are assembled onto vectors.In particular, the genes hpcH (CAA87759), hpcG (CAA57202), enr(YP_430895) and lysN ( ) genes encoding2-oxo-4-hydroxy-7-aminoheptanoate aldolase,2-oxo-4-hydroxy-7-aminoheptanoate dehydratase,2-oxo-7-aminohept-3-enoate reductase and 2-oxo-7-aminoheptanoateaminotransferase, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany), under the control of the PA1/lacOpromoter. The plasmid is transformed into E. coli strain MG1655 toexpress the proteins and enzymes required for HMDA synthesis from4-aminobutanal. E. coli naturally encodes two lysine decarboxylaseenzymes which convert homolysine to HMDA.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of HMDA pathwaygenes is corroborated using methods well known in the art fordetermining polypeptide expression or enzymatic activity, including forexample, Northern blots, PCR amplification of mRNA and immunoblotting.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individually activities. The ability of the engineeredE. coli strain to produce HMDA through this pathway is confirmed usingHPLC, gas chromatography-mass spectrometry (GCMS) or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional HMDA synthesis pathwayfrom 4-aminobutanal are further augmented by optimization for efficientutilization of the pathway. Briefly, the engineered strain is assessedto determine whether any of the exogenous genes are expressed at a ratelimiting level. Expression is increased for any enzymes expressed at lowlevels that can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

After successful demonstration of enhanced HMDA production via theactivities of the exogenous enzymes, the genes encoding these enzymesare inserted into the chromosome of a wild type E. coli host usingmethods known in the art. Such methods include, for example, sequentialsingle crossover (Gay et al., J. Bacteriol. 3:153 (1983)). and Red/ETmethods from GeneBridges (Zhang et al., European Patent Application No.01117 (2001))). Chromosomal insertion provides several advantages over aplasmid-based system, including greater stability and the ability toco-localize expression of pathway genes.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of HMDA. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of HMDA.Adaptive evolution also can be used to generate better producers of, forexample, the 2-oxo-4-hydroxy-7-aminoheptanoate intermediate or the HMDAproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the HMDA producer to further increaseproduction.

For large-scale production of HMDA, the above HMDA pathway-containingorganism is cultured in a fermenter using a medium known in the art tosupport growth of the organism under anaerobic conditions. Fermentationsare performed in either a batch, fed-batch or continuous manner.Anaerobic conditions are maintained by first sparging the medium withnitrogen and then sealing culture vessel (e.g., flasks can be sealedwith a septum and crimp-cap). Microaerobic conditions also can beutilized by providing a small hole for limited aeration. The pH of themedium is maintained at a pH of 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids,Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Example XXV Pathways for Production of 6-Aminocaproate from Glutamate,Glutaryl-CoA,Homolysine, or 2-Amino-7-oxosubarate

Novel pathways for producing 6-aminocaproate (6-ACA) and relatedproducts are described herein. The candidate enzymes, and associatedrisks of implementation are discussed in Example XXVI below.

This invention is directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyze 6-ACAproduction. Successfully engineering these pathways entails identifyingan appropriate set of enzymes with sufficient activity and specificity,cloning their corresponding genes into a production host, optimizing theexpression of these genes in the production host, optimizingfermentation conditions, and assaying for product formation followingfermentation.

6-ACA can be produced from glutamate as a starting molecule. Glutamateis transformed to 6-aminopimeloyl-CoA as described previously (FIG. 20,Steps A-E). Removal of the CoA moiety of 6-Aminopimeloyl-CoA by a CoAhydrolase, transferase or ligase yields 2-aminopimelate (Step I of FIG.20). Decarboxylation of this product yields 6-ACA (Step J of FIG. 20).

6-ACA can also be produced from glutaryl-CoA as a starting molecule. Inthe disclosed pathway to 6-ACA, similar to the HMDA pathway describedabove, glutaryl-CoA is first condensed with acetyl-CoA by abeta-ketothiolase to form 3-oxopimeloyl-CoA (Step A of FIG. 21). The CoAmoiety of 3-oxopimeloyl-CoA is removed by a CoA hydrolase, transferaseand ligase (Step B of FIG. 21). Then 3-oxopimelate is transaminated to3-aminopimelate (Step J of FIG. 21). 3-Aminopimelate can be converted to2-aminopimelate by a 2,3-aminomutase enzyme (Step T of FIG. 21).Aminopimelate can then be decarboxylated to form 6-aminocaproic acid(Step AA of FIG. 21).

Homolysine is also an attractive precursor to 6-aminocaproate (6-ACA)production. Although homolysine is a potentially valuable precursor, itis not a known metabolic intermediate of any organism. Under aerobicconditions, oxidation of homolysine by a lysine 2-monooxygenase yields6-aminohexanamide, which is readily hydrolyzed to 6-ACA in dilute acidor basic solution (FIG. 23).

6-ACA can also be produced from 2-amino-7-oxosubarate as a startingmolecule (FIG. 26). 2-Amino-7-oxosubarate is not known to be a naturallyoccurring metabolite. An exemplary route for synthesizing2-amino-7-oxosubarate is shown in FIG. 27. The pathway originates withglutamate-5-semialdehyde, a metabolite naturally formed during ornithinebiosynthesis. 2-Amino-7-oxosubarate is then synthesized in threeenzymatic steps. In the first step, glutamate-5-semialdehyde iscondensed with pyruvate by an aldolase (FIG. 27, Step A). The product,2-amino-5-hydroxy-7-oxosubarate is subsequently dehydrated and theresulting alkene is reduced to form 2-amino-7-oxosubarate (FIG. 27,Steps B/C). In one proposed route, 2-amino-7-oxosubarate isdecarboxylated to form 2-amino-7-oxoheptanoate (Step A of FIG. 26). Thealdehyde of 2-amino-7-oxoheptanoate is oxidized by an oxidoreductase toform 2-aminopimelate (Step D of FIG. 26). 6-ACA is the decarboxylationproduct of 2-aminopimelate (Step E of FIG. 26). Alternately, the2-amino-7-oxoheptanoate intermediate is decarboxylated to form6-aminohexanal (Step B of FIG. 26), which is transaminated to 6-ACA(Step F of FIG. 26). In a third proposed route, the 2-amino acid groupof 2-amino-7-oxosubarate is decarboxylated, yielding2-oxo-7-aminoheptanoate (Step I of FIG. 26). This product can then befurther decarboxylated to 6-aminohexanal (Step G of FIG. 26). Finally,6-aminohexanal is transaminated to 6-ACA (Step F of FIG. 26).

Example XXVI Enzyme Classification System for Production ofHexamethylenediamine and 6-Aminocaproic acid

This example describes the enzyme classification system for theexemplary pathways described in Examples XXIV and XXV for production ofhexamethylenediamine or 6-aminocaproate.

All transformations depicted in FIGS. 20-23 and 26 fall into the generalcategories of transformations shown in Table 11. Below is described anumber of biochemically characterized genes in each category.Specifically listed are genes that can be applied to catalyze theappropriate transformations in FIGS. 20-23 and 26 when properly clonedand expressed.

Table 11 shows the enzyme types useful to convert common centralmetabolic intermediates into 6-aminocaproate and hexamethylenediamine.The first three digits of each label correspond to the first threeEnzyme Commission number digits which denote the general type oftransformation independent of substrate specificity.

TABLE 11 LABEL FUNCTION 1.1.1.a Oxidoreductase (oxo to alcohol)1.13.12.a Monooxygenase (O₂ incorporating) 1.2.1.a Oxidoreductase(aldehyde to acid) 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.2.1.dOxidoreductase (phosphonate reductase) 1.2.1.e Acid reductase 1.3.1.aOxidoreductase (alkene to alkane) 1.4.1.a Oxidoreductase (aminating)2.3.1.b Acyltransferase (beta-ketothiolase) 2.6.1.a Aminotransferase2.7.2.a Phosphotransferase (carboxy acceptor) 2.8.3.a Coenzyme-Atransferase 3.1.2.a CoA hydrolase 4.1.1.a Carboxy-lyase 4.1.2.aAldehyde-lyase 4.2.1.a Hydro-lyase 5.4.3.a Aminomutase 6.2.1.aAcid-thiol ligase

1.1.1.a Oxidoreductase (Oxo to Alcohol)—

The reduction of 3-oxo-6-aminopimeloyl-CoA to3-hydroxy-6-aminopimeloyl-CoA is catalyzed by a 3-oxoacyl-CoAdehydrogenase (FIG. 20, Step C). Such enzymes convert 3-oxoacyl-CoAmolecules into 3-hydroxyacyl-CoA molecules and are often involved infatty acid beta-oxidation or phenylacetate catabolism. For example,subunits of two fatty acid oxidation complexes in E. coli, encoded byfadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock etal., Methods Enzymol. 71 Pt C:403-411 (1981)). Furthermore, the geneproducts encoded by phaC in Pseudomonas putida U (Olivera et al., Proc.Natl. Acad. Sci U.S.A 95:6419-6424 (1998)) and paaC in Pseudomonasfluorescens ST (Di Arch et al., Microbiol 188:117-125 (2007)) catalyzethe reverse reaction of step B in FIG. 10, that is, the oxidation of3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism ofphenylacetate or styrene. Note that the reactions catalyzed by suchenzymes are reversible. In addition, given the proximity in E. coli ofpaaH to other genes in the phenylacetate degradation operon (Nogales etal., Microbiology 153:357-365 (2007)) and the fact that paaH mutantscannot grow on phenylacetate (Ismail et al., Eur. J Biochem.270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodesa 3-hydroxyacyl-CoA dehydrogenase.

GenBank Gene name GI# Accession # Organism fadB 119811 P21177.2Escherichia coli fadJ 3334437 P77399.1 Escherichia coli paaH 16129356NP_415913.1 Escherichia coli phaC 26990000 NP_745425.1 Pseudomonasputida paaC 106636095 ABF82235.1 Pseudomonas fluorescens

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoAmolecules to their corresponding 3-hydroxyacyl-CoA molecules include3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridiumacetobutylicum, encoded by hbd, has been cloned and functionallyexpressed in E. coli (Youngleson et al., J Bacteriol. 171:6800-6807(1989)). Additional gene candidates include Hbd1 (C-terminal domain) andHbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer et al., FEBSLett. 21:351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al., JBiol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstratedto reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloearamigera (Ploux et al., Eur. J Biochem. 174:177-182 (1988)) and phaBfrom Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61:297-309(2006)).) The former gene candidate is NADPH-dependent, its nucleotidesequence has been determined (Peoples et al., Mol. Microbiol 3:349-357(1989)) and the gene has been expressed in E. coli. Substratespecificity studies on the gene led to the conclusion that it couldaccept 3-oxopropionyl-CoA as an alternate substrate (Peoples et al.,Mol. Microbiol 3:349-357 (1989)).

GenBank Gene name GI# Accession # Organism hbd 18266893 P52041.2Clostridium acetobutylicum Hbd2 146348271 EDK34807.1 Clostridiumkluyveri Hbd1 146345976 EDK32512.1 Clostridium kluyveri HSD17B10 3183024O02691.3 Bos taurus phaB 77464321 YP_353825.1 Rhodobacter sphaeroidesphbB 130017 P23238.1 Zoogloea ramigera

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science.318:1782-1786 (2007).

GenBank Gene name GI# Accession # Organism hbd 15895965 NP_349314.1Clostridium acetobutylicum hbd 20162442 AAM14586.1 Clostridiumbeijerinckii Msed_1423 146304189 YP_001191505 Metallosphaera sedulaMsed_0399 146303184 YP_001190500 Metallosphaera sedula Msed_0389146303174 YP_001190490 Metallosphaera sedula Msed_1993 146304741YP_001192057 Metallosphaera sedula

1.13.12.a Monooxygenase (O₂ Incorporating)—

An 02-incorporating monooxygenase is required to convert homolysine to6-aminohexanamide (Step A of FIG. 23). The lysine 2-monooxygenase (EC1.13.12.2) from Pseudomonas fluorescens reacts with homolysine as asubstrate (Nakazawa et al., J Biol. Chem. 247:3439-3444 (1972)). Theenzyme from P. putida has been characterized biochemically and the genehas been identified (Karyakin et al., Prikladnaya Biokhimiya iMikrobiologiya 27:825-832 (1991)). Genes encoding lysine 2-monooxygenaseenzymes in P. fluorescens (eval=0.0, 90% identity), Streptomycescoelicolor (eval=0.0, 58% identity), Rhodococcus jostii (eval=0.0, 56%identity) and others were identified by protein sequence homology to theP. putida enzyme.

GenBank Gene name GI# Accession # Organism davB 193805882 BAG54787.1Pseudomonas putida pfl_5670 68347255 AAY94861.1 Pseudomonas fluorescensSCO1454 7209214 CAB76876.1 Streptomyces coelicolor RHA1_ro03531110820050 ABG95334.1 Rhodococcus jostii

1.2.1.a Oxidoreductase (Aldehyde to Acid)

Two transformations in FIG. 26 require conversion of an aldehyde to anacid: conversion of 2-amino-7-oxoheptanoate to 2-aminopimelate (Step D)and 6-aminohexanal to 6-aminocaproate (Step F). Such reactions arecatalyzed by NAD(P)+-dependent oxidoreductases that convert aldehydes toacids in the EC class 1.2.1. A candidate enzyme is the NAD+-dependentaldehyde dehydrogenase (EC 1.2.1.3). Two aldehyde dehydrogenases foundin human liver, ALDH-1 and ALDH-2, have broad substrate ranges for avariety of aliphatic, aromatic and polycyclic aldehydes (Klyosov et al.,Biochemistry 35:4457-4467 (1996)). Active ALDH-2 has been efficientlyexpressed in E. coli using the GroEL proteins as chaperonins (Lee etal., Biochem. Biophys. Res. Commun. 298:216-224 (2002)). The ratmitochondrial aldehyde dehydrogenase also has a broad substrate rangethat includes the enoyl-aldehyde crotonaldehyde (Siew et al., Arch.Biochem. Biophys. 176:638-649 (1976)). The E. coli gene astD alsoencodes an NAD+-dependent aldehyde dehydrogenase that converts succinicsemialdehyde to succinate (Kuznetsova et al., FEMS Microbiol Rev29:263-279 (2005)).

Gene name GI# GenBank Accession # Organism ALDH-2 118504 P05091.2 Homosapiens ALDH-2 14192933 NP_115792.1 Rattus norvegicus astD 3913108P76217.1 Escherichia coli

1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde)—

Reductive de-acylation of 3-oxopimeloyl-CoA (FIG. 21, Step I),5-aminopimeloyl-CoA (FIG. 21, Step L) and 6-aminopimeloyl-CoA (FIG. 21,Step Y) to their corresponding aldehydes is catalyzed by enzymes in theEC class 1.2.1. Exemplary acyl-CoA dehydrogenases that reduce anacyl-CoA to its corresponding aldehyde include the fatty acid acyl-CoAreductase enzymes of Acinetobacter calcoaceticus (Reiser et al., Journalof Bacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishigeet al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- andNADP-dependent succinate semialdehyde dehydrogenase encoded by the sucDgene in Clostridium kluyveri (Sohling et al., J Bacteriol. 178:871-880(1996); and Sohling et al., J Bacteriol 178:871-80 (1996)). SucD of P.gingivalis is another succinate semialdehyde dehydrogenase (Takahashi etal., J. Bacteriol. 182:4704-4710 (2000)). The enzyme acylatingacetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yetanother as it has been demonstrated to oxidize and acylate acetaldehyde,propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde(Powlowski et al., J Bacteriol. 175:377-385 (1993)). In addition toreducing acetyl-CoA to ethanol, the enzyme encoded by adhE inLeuconostoc mesenteroides has been shown to oxidize the branched chaincompound isobutyraldehyde to isobutyryl-CoA (Koo et al., BiotechnolLett. 27:505-510 (2005)).

GenBank Gene name GI# Accession # Organism acr1 50086359 YP_047869.1Acinetobacter calcoaceticus acr1 1684886 AAC45217 Acinetobacter baylyiacr1 18857901 BAB85476.1 Acinetobacter sp. Strain M-1 sucD 172046062P38947.1 Clostridium kluyveri sucD 34540484 NP_904963.1 Porphyromonasgingivalis bphG 425213 BAA03892.1 Pseudomonas sp

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archael bacteria (Berg et al., Science. 318:1782-1786(2007); and Thauer et al., Science. 318:1732-1733 (2007)). The enzymeutilizes NADPH as a cofactor and has been characterized inMetallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol.188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410(2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula(Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al.,Science. 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductasefrom Sulfolobus tokodaii was cloned and heterologously expressed in E.coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme hasalso been shown to catalyze the conversion of methylmalonyl-CoA to itscorresponding aldehyde (WO/2007/141208). Although the aldehydedehydrogenase functionality of these enzymes is similar to thebifunctional dehydrogenase from Chloroflexus aurantiacus, there islittle sequence similarity. Both malonyl-CoA reductase enzyme candidateshave high sequence similarity to aspartate-semialdehyde dehydrogenase,an enzyme catalyzing the reduction and concurrent dephosphorylation ofaspartyl-4-phosphate to aspartate semialdehyde. Additional genecandidates can be found by sequence homology to proteins in otherorganisms including Sulfolobus solfataricus and Sulfolobusacidocaldarius. Yet another candidate for CoA-acylating aldehydedehydrogenase is the ald gene from Clostridium beijerinckii (Toth etal., Appl Environ. Microbiol 65:4973-4980 (1999)). This enzyme has beenreported to reduce acetyl-CoA and butyryl-CoA to their correspondingaldehydes. This gene is very similar to eutE that encodes acetaldehydedehydrogenase of Salmonella typhimurium and E. coli (Toth et al., ApplEnviron. Microbiol 65:4973-4980 (1999)).

GenBank Gene name GI# Accession # Organism Msed_0709 146303492YP_001190808.1 Metallosphaera sedula mcr 15922498 NP_378167.1 Sulfolobustokodaii asd-2 15898958 NP_343563.1 Sulfolobus solfataricus Saci_237070608071 YP_256941.1 Sulfolobus acidocaldarius Ald 49473535 AAT66436Clostridium beijerinckii eutE 687645 AAA80209 Salmonella typhimuriumeutE 2498347 P77445 Escherichia coli

1.2.1.d Oxidoreductase (Phosphonate Reductase)—

The reduction of a phosphonic acid to its corresponding aldehyde iscatalyzed by an oxidoreductase in the EC class 1.2.1. Steps G, N and Xin FIG. 21 require such an enzyme for the reduction of5-oxopimeloyl-phosphonate, 5-aminopimeloylphosphonate and6-aminopimeloylphosphonate to their corresponding aldehydes. Thesereactions are not catalyzed by known enzymes. A similar reaction iscatalyzed by aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11):the NADPH-dependent reduction of 4-aspartyl phosphate toaspartate-4-semialdehyde. ASD participates in amino acid biosynthesisand recently has been studied as an antimicrobial target (Hadfield etal., Biochemistry 40:14475-14483 (2001)). The E. coli ASD structure hasbeen solved (Hadfield et al., J Mol. Biol. 289:991-1002 (1999)) and theenzyme has been shown to accept the alternate substratebeta-3-methylaspartyl phosphate (Shames, et al., J Biol. Chem.259:15331-15339 (1984)). The Haemophilus influenzae enzyme has been thesubject of enzyme engineering studies to alter substrate bindingaffinities at the active site (Blanco et al., Crystallogr. 60:1388-1395(2004)). Other ASD candidates are found in Mycobacterium tuberculosis(Shafiani et al., J Appl Microbiol 98:832-838 (2005)), Methanococcusjannaschii Faehnle et al. J Mol. 353:1055-1068 (2005)) and theinfectious microorganisms Vibrio cholera and Heliobacter pylori (Mooreet al., Protein Expr. Purif. 25:189-194 (2002)). Acetylglutamylphosphatereductase (EC 1.2.1.38) is a related enzyme that naturally reducesacetylglutamylphosphate to acetylglutamate-5-semialdehyde. Genesencoding this enzyme are found in S. cerevisiae (Pauwels et al., Eur. JBiochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly et al.,Microbiology 140 (Pt 5):1023-1025 (1994)) and other organisms.

GenBank Gene name GI# Accession # Organism asd 16131307 NP_417891.1Escherichia coli asd 68249223 YP_248335.1 Haemophilus influenzae asd1899206 AAB49996 Mycobacterium tuberculosis VC2036 15642038 NP_231670Vibrio cholera asd 210135348 YP_002301787.1 Heliobacter pylori ARG5,66320913 NP_010992.1 Saccharomyces cerevisiae argC 16078184 NP_389001.1Bacillus subtilis

Other exemplary phosphonate reductase enzymes include glyceraldehyde3-phosphate dehydrogenase which converts glyceraldehyde-3-phosphate intoD-glycerate 1,3-bisphosphate (e.g., E. coli gapA (Branlant et al., Eur.J. Biochem. 150:61-66 (1985)).23)), N-acetyl-gamma-glutamyl-phosphatereductase which converts N-acetyl-L-glutamate-5-semialdehyde intoN-acetyl-L-glutamyl-5-phosphate (e.g., E. coli argC (Parsot et al.,Gene. 68:275-283 (1988)), and glutamate-5-semialdehyde dehydrogenasewhich converts L-glutamate-5-semialdehyde into L-glutamyl-5-phospate(e.g., E. coli proA (Smith et al., J. Bacteriol. 157:545-551 (1984))).Genes encoding glutamate-5-semialdehyde dehydrogenase enzymes fromSalmonella typhimurium (Mahan et al., J Bacteriol. 156:1249-1262 (1983))and Campylobacter jejuni (Louie et al., Mol. Gen. Genet. 240:29-35(1993)) were cloned and expressed in E. coli.

GenBank Gene name GI# Accession # Organism gapA 71159358 P0A9B2.2Escherichia coli argC 16131796 NP_418393.1 Escherichia coli proA16128229 NP_414778.1 Escherichia coli proA 16763704 NP_459319.1Salmonella typhimurium proA 9087222 P53000.2 Campylobacter jejuni

1.2.1.e Acid Reductase—

Several transformations in FIG. 21 require the conversion of an acid toan aldehyde (FIG. 21, Steps C, O, W). Such a transformation isthermodynamically unfavorable and typically requires energy-richcofactors and multiple enzymatic steps. For example, in butanolbiosynthesis conversion of butyrate to butyraldehyde is catalyzed byactivation of butyrate to its corresponding acyl-CoA by a CoAtransferase or ligase, followed by reduction to butyraldehyde by aCoA-dependent aldehyde dehydrogenase. Alternately, an acid can beactivated to an acyl-phosphate and subsequently reduced by a phosphatereductase. Direct conversion of the acid to aldehyde by a single enzymeis catalyzed by an enzyme in the 1.2.1 family. Exemplary enzymes thatcatalyze these transformations include carboxylic acid reductase,alpha-aminoadipate reductase and retinoic acid reductase.

Carboxylic acid reductase, found in Nocardia iowensis, catalyzes themagnesium, ATP and NADPH-dependent reduction of carboxylic acids totheir corresponding aldehydes (Venkitasubramanian et al., J Biol. Chem.282:478-485 (2007)). This enzyme, encoded by car, was cloned andfunctionally expressed in E. coli (Venkitasubramanian et al., J Biol.Chem. 282:478-485 (2007)). Expression of the npt gene product improvedactivity of the enzyme via post-transcriptional modification. The nptgene encodes a specific phosphopantetheine transferase (PPTase) thatconverts the inactive apo-enzyme to the active holo-enzyme. The naturalsubstrate of this enzyme is vanillic acid and the enzyme exhibits broadacceptance of aromatic and aliphatic substrates (Venkitasubramanian etal. “Biocatalytic Reduction of Carboxylic Acids: Mechanism andApplications” Chapter 15 in Biocatalysis in the Pharmaceutical andBiotechnology Industries, ed. R. N. Patel, CRC Press LLC, Boca Raton,Fla. (2006)).

GenBank Gene name GI# Accession # Organism car 40796035 AAR91681.1Nocardia iowensis (sp. NRRL 5646) npt 114848891 ABI83656.1 Nocardiaiowensis (sp. NRRL 5646)

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J Biol.Chem 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTasehas not been identified to date and no high-confidence hits wereidentified by sequence comparison homology searching. Directed evolutionor other enzyme engineering methods may be required to enhancereactivity with the substrates in FIG. 21.

GenBank Gene name GI# Accession # Organism LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

1.3.1.a Oxidoreductase (Alkene to Alkane)—

Three transformations fall into the category of oxidoreductases thatreduce an alkene to an alkane (EC 1.3.1.-). The conversion of6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA (FIG. 20, StepE), 2-oxo-7-aminohept-3-onoate to 2-oxo-7-aminoheptanoate (FIG. 22, StepC) and 2-amino-5-ene-7-oxosubarate to 2-amino-7-oxosubarate (FIG. 27,Step C) are catalyzed by a 2-enoate reductase. 2-Enoate reductaseenzymes are known to catalyze the NAD(P)H-dependent reduction of a widevariety of α, β-unsaturated carboxylic acids and aldehydes (Rohdich, etal., J Biol. Chem. 276:5779-5787 (2001)). In the recently publishedgenome sequence of C. kluyveri, 9 coding sequences for enoate reductaseswere reported, out of which one has been characterized (Seedorf et al.,Proc. Natl. Acad. Sci U.S.A 105:2128-2133 (2008)). The enr genes fromboth C. tyrobutyricum and M. thermoaceticum have been cloned andsequenced and show 59% identity to each other. The former gene is alsofound to have approximately 75% similarity to the characterized gene inC. kluyveri (Giesel et al., Arch. Microbiol 135:51-57 (1983)). It hasbeen reported based on these sequence results that enr is very similarto the dienoyl CoA reductase in E. coli (fadH) (Rohdich, et al., J Biol.Chem. 276:5779-5787 (2001)). The Moorella thermoacetica (formerly C.thermoaceticum) enr gene has also been expressed in a catalyticallyactive form in E. coli (Ohdich, et al., J Biol. Chem. 276:5779-5787(2001)).

GenBank Gene name GI# Accession # Organism enr 169405742 ACA54153.1Clostridium botulinum A3 str enr 2765041 CAA71086.1 Clostridiumtyrobutyricum enr 3402834 CAA76083.1 Clostridium kluyveri enr 83590886YP_430895.1 Moorella thermoacetica fadH 16130976 NP_417552.1 Escherichiacoli

Another candidate 2-enoate reductase is maleylacetate reductase (MAR),an enzyme catalyzing the reduction of 2-maleylacetate(4-oxohex-2-enedioate) to 3-oxoadipate. MAR enzymes naturallyparticipate in aromatic degradation pathways (Camara et al., JBacteriol. (2009); Huang et al., Appl Environ. Microbiol 72:7238-7245(2006)); Kaschabek et al., J Bacteriol. 177:320-325 (1995) and Kaschabeket al., J Bacteriol. 175:6075-6081 (1993)). The enzyme activity wasidentified and characterized in Pseudomonas sp. strain B13 (Kaschabek etal., J Bacteriol 177:320-325 (1995); and Kaschabek et al., J Bacteriol175:6075-6081 (1993)), and the coding gene was cloned and sequenced(Kasberg et al., J Bacteriol. 179:3801-3803 (1997)). Additional MAR genecandidates include cicE gene from Pseudomonas sp. strain B13 (Kasberg etal., J Bacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcusopacus (Seibert et al., J Bacteriol 180:3503-3508 (1998)), the macA genefrom Ralstonia eutropha (also known as Cupriavidus necator) (Seibert etal., Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha(Seibert et al., J Bacteriol. 175:6745-6754 (1993)) and NCgI1112 inCorynebacterium glutamicum (Huang et al., Appl Environ. Microbiol72:7238-7245 (2006)). A MAR in Pseudomonas reinekei MT1, encoded byccaD, was recently identified and the nucleotide sequence is availableunder the DBJ/EMBL GenBank accession number EF159980 (Camara et al., JBacteriol. (2009)).

GenBank Gene name GI# Accession # Organism clcE 3913241 O30847.1Pseudomonas sp. strain B13 macA 7387876 O84992.1 Rhodococcus opacus macA5916089 AAD55886 Cupriavidus necator tfdFII 1747424 AAC44727.1 Ralstoniaeutropha JMP134 NCgl1112 19552383 NP_600385 Corynebacterium glutamicumccaD 134133940 ABO61029.1 Pseudomonas reinekei MT1

Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing thereduction of 6-amino-7-carboxyhept-2-enoyl-CoA to 6-aminopimeloyl-CoA(FIG. 20, Step E). One exemplary enoyl-CoA reductase is the gene productof bcd from C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311(2008)); and Boynton et al., J Bacteria 178:3015-3024 (1996)), whichnaturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA.Activity of this enzyme can be enhanced by expressing bcd in conjunctionwith expression of the C. acetobutylicum etfAB genes, which encode anelectron transfer flavoprotein. An additional candidate for theenoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase fromE. gracilis (Hoffmeister, et al., J Biol. Chem. 280:4329-4338 (2005)). Aconstruct derived from this sequence following the removal of itsmitochondrial targeting leader sequence was cloned in E. coli resultingin an active enzyme (Hoffmeister, et al., J Biol. Chem. 280:4329-4338(2005)). This approach is well known to those skilled in the art ofexpressing eukaryotic genes, particularly those with leader sequencesthat may target the gene product to a specific intracellularcompartment, in prokaryotic organisms. A close homolog of this gene,TDE0597 from the prokaryote Treponema denticola, represents a thirdenoyl-CoA reductase which has been cloned and expressed in E. coli(Tucci et al., Febs Letters 581:1561-1566 (2007)).

GenBank Gene name GI# Accession # Organism bcd 15895968 NP_349317.1Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridiumacetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum TER62287512 Q5EU90.1 Euglena gracilis TDE0597 42526113 NP_971211.1Treponema denticola

Additional enoyl-CoA reductase enzyme candidates are found in organismsthat degrade aromatic compounds. Rhodopseudomonas palustris, a modelorganism for benzoate degradation, has the enzymatic capability todegrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes inthe pim operon, pimC and pimD, bear sequence homology to C.acetobutylicum bcd and are predicted to encode a flavin-containingpimeloyl-CoA dehydrogenase (Harrison et al., Microbiology 151:727-736(2005)). The genome of nitrogen-fixing soybean symbiont Bradyrhizobiumjaponicum also contains a pim operon composed of genes with highsequence similarity to pimC and pimD of R. palustris (Harrison et al.,Microbiology 151:727-736 (2005)).

GenBank Gene name GI# Accession # Organism pimC 39650632 CAE29155Rhodopseudomonas palustris pimD 39650631 CAE29154 Rhodopseudomonaspalustris pimC 27356102 BAC53083 Bradyrhizobium japonicum pimD 27356101BAC53082 Bradyrhizobium japonicum

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase(EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hinderedtrans-enoyl-CoA substrates. This enzyme participates in branched-chainfatty acid synthesis in the nematode Ascarius suum and is capable ofreducing a variety of linear and branched chain substrates including2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA andpentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993))). Twoisoforms of the enzyme, encoded by genes acad1 and acad, have beencharacterized.

Gene name GI# GenBank Accession # Organism acad1 2407655 AAC48316.1Ascarius suum acad 347404 AAA16096.1 Ascarius suum

1.4.1.a Oxidoreductase (Aminating)—

Several reactions in FIGS. 20-23 require the conversion of ketones oraldehydes to amine groups. Such a transformation can be accomplished byaminating oxidoreductases in the EC class 1.4.1. Enzymes in this ECclass catalyze the oxidative deamination of amino groups with NAD+ orNADP+ as acceptor, and the reactions are typically reversible.

In Step D of FIG. 22 the 2-oxoacid 2-oxo-7-aminoheptanoate is convertedto homolysine, a molecule resembling an amino acid (FIG. 22, Step D;FIG. 26, Step J). The conversion of 2-amino-7-oxosubarate to2,7-diaminosubarate (Step K of FIG. 26) is a similar transformation.Exemplary enzymes for catalyzing these reactions include glutamatedehydrogenase (EC 1.4.1.2), leucine dehydrogenase (EC 1.4.1.9), andaspartate dehydrogenase (EC 1.4.1.21). The gdhA gene product fromEscherichia coli (Korber, et al., J Mol. Biol. 234:1270-1273. (1993)),gdh from Thermotoga maritime (Kort et al., Extremophiles 1:52-60. 1997);Lebbink et al., J Mol. Biol. 280:287-296 (1998) and Lebbink et al., JMol. Biol. 289:357-369 (1999))), and gdhA1 from Halobacterium salinarum(Ingoldsby et al., Gene 349:237-244 (2005)) catalyze the reversibleconversion of glutamate to 2-oxoglutarate and ammonia, while favoringNADP(H), NAD(H), or both, respectively. The ldh gene of Bacillus cereusencodes the LeuDH protein that has a wide of range of substratesincluding leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge etal., Biotechnol Bioeng. 68:557-562 (2000)); and Stoyan et al., JBiotechnol 54:77-80 (1997)). The nadX gene from Thermotoga maritimaencoding for the aspartate dehydrogenase is involved in the biosynthesisof NAD (Yang et al., J Biol. Chem. 278:8804-8808 (2003)).

GenBank Gene name GI# Accession # Organism gdhA 118547 P00370Escherichia coli gdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827NP_279651.1 Halobacterium salinarum ldh 61222614 P0A393 Bacillus cereusnadX 15644391 NP_229443.1 Thermotoga maritima

Two reactions entail conversion of 3-oxoacids to 3-amino acids:3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate (FIG. 21, Step E),3-oxopimelate to 3-aminopimelate (FIG. 21, Step J) and3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoate (FIG. 21, Step AB).An enzyme that reacts with 3-oxoacids is 3,5-diaminohexanoatedehydrogenase (EC 1.4.1.11), an enzyme found in organisms that fermentlysine. The gene encoding this enzyme, kdd, was recently identified inFusobacterium nucleatum (Kreimeyer et al., J Biol. Chem. 282:7191-7197(2007)). The enzyme has been purified and characterized in otherorganisms (Baker et al., Chem. 247:7724-7734 (1972)); and Baker et al.,Biochemistr. 13:292-299 (1974)) but the genes associated with theseenzymes are not known. Candidates in Myxococcus xanthus, Porphyromonasgingivalis W83 and other sequenced organisms can be inferred by sequencehomology.

GenBank Gene name GI# Accession # Organism kdd 19713113 AAL93966.1Fusobacterium nucleatum mxan_4391 108462082 ABF87267.1 Myxococcusxanthus pg_1069 34397119 AAQ66183.1 Porphyromonas gingivalis

The conversions of 2-amino-7-oxoheptanoate to homolysine (FIG. 20, StepG; FIG. 21, Step Q; FIG. 26, Step M), 3-oxo-1-carboxyheptanal to3-oxo-7-aminoheptanoate (FIG. 21, Step D) 3-amino-7-oxoheptanoate to3,7-diaminoheptanoate (FIG. 21, Step Z) and 6-aminohexanal to HMDA (FIG.26, Step C; FIG. 22, Step G) are catalyzed by aminating oxidoreductasesthat transform aldehydes to their corresponding primary amines. Anenzyme that catalyzes a similar reaction is lysine 6-dehydrogenase (EC1.4.1.18), encoded by the lysDH genes. This enzyme catalyzes thereversible oxidative deamination of the 6-amino group of L-lysine toform 2-aminoadipate-6-semialdehyde (Misono et al., J Bacteriol.150:398-401 (1982)). Exemplary enzyme candidates are found inGeobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., JBiochem 106:76-80 (1989); and Misono et al., J Bacteriol. 150:398-401(1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMPRep. 41:790-795 (2008)).

GenBank Gene name GI# Accession # Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

2.3.1.b Acyltransferase (Beta-Ketothiolase)—

In Step A of FIG. 21, Glutaryl-CoA and acetyl-CoA are condensed to form3-oxopimeloyl-CoA by oxopimeloyl-CoA:glutaryl-CoA acyltransferase, abeta-ketothiolase (EC 2.3.1.16). An enzyme catalyzing thistransformation is found in Ralstonia eutropha (formerly known asAlcaligenes eutrophus), encoded by genes bktB and bktC (Haywood et al.,FEMS Microbiology Letters 52:91-96 (1988); and Slater et al., Bacteriol.180:1979-1987 (1998)). The sequence of the BktB protein is known;however, the sequence of the BktC protein has not been reported. The pimoperon of Rhodopseudomonas palustris also encodes a beta-ketothiolase,encoded by pimB, predicted to catalyze this transformation in thedegradative direction during benzoyl-CoA degradation (Harrison et al.,Microbiology 151:727-736 (2005)). A beta-ketothiolase enzyme candidatein S. aciditrophicus was identified by sequence homology to bktB (43%identity, evalue=1e-93).

GenBank Gene name GI# Accession # Organism bktB 11386745 YP_725948Ralstonia eutropha pimB 39650633 CAE29156 Rhodopseudomonas palustrissyn_02642 85860483 YP_462685.1 Syntrophus aciditrophicus

Beta-ketothiolase enzymes catalyzing the formation of beta-ketovaleratefrom acetyl-CoA and propionyl-CoA may also be able to catalyze theformation of 3-oxopimeloyl-CoA. Zoogloea ramigera possesses twoketothiolases that can form β-ketovaleryl-CoA from propionyl-CoA andacetyl-CoA and R. eutropha has a β-oxidation ketothiolase that is alsocapable of catalyzing this transformation (Gruys et al., U.S. Pat. No.5,958,745 (1999)). The sequences of these genes or their translatedproteins have not been reported, but several candidates in R. eutropha,Z. ramigera, or other organisms can be identified based on sequencehomology to bktB from R. eutropha. These include:

GenBank Gene name GI# Accession # Organism phaA 113867452 YP_725941.1Ralstonia eutropha h16_A1713 113867716 YP_726205.1 Ralstonia eutrophapcaF 116694155 YP_728366.1 Ralstonia eutropha h16_B1369 116695312YP_840888.1 Ralstonia eutropha h16_A0170 113866201 YP_724690.1 Ralstoniaeutropha h16_A0462 113866491 YP_724980.1 Ralstonia eutropha h16_A1528113867539 YP_726028.1 Ralstonia eutropha h16_B0381 116694334 YP_728545.1Ralstonia eutropha h16_B0662 116694613 YP_728824.1 Ralstonia eutrophah16_B0759 116694710 YP_728921.1 Ralstonia eutropha h16_B0668 116694619YP_728830.1 Ralstonia eutropha h16_A1720 113867723 YP_726212.1 Ralstoniaeutropha h16_A1887 113867867 YP_726356.1 Ralstonia eutropha phbA 135759P07097.4 Zoogloea ramigera bktB 194289475 YP_002005382.1 Cupriavidustaiwanensis Rmet_1362 94310304 YP_583514.1 Ralstonia metalliduransBphy_0975 186475740 YP_001857210.1 Burkholderia phymatum

Additional candidates include beta-ketothiolases that are known toconvert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9).Exemplary acetoacetyl-CoA thiolase enzymes include the gene products ofatoB from E. coli (Martin et al., Nat. Biotechnol 21:796-802 (2003)),thlA and thlB from C. acetobutylicum (Hanai et al., Appl EnvironMicrobiol 73:7814-7818 (2007)); and Winzer et al., J Mol. MicrobiolBiotechnol 2:531-541 (2000)), and ERG10 from S. cerevisiae (Hiser, etal., J. Biol. Chem. 269:31383-31389 (1994)).

GenBank Gene name GI# Accession # Organism atoB 16130161 NP_416728Escherichia coli thlA 15896127 NP_349476.1 Clostridium acetobutylicumthlB 15004782 NP_149242.1 Clostridium acetobutylicum ERG10 6325229NP_015297 Saccharomyces cerevisiae

Beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoAthiolase, converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA,and is a key enzyme of the beta-ketoadipate pathway for aromaticcompound degradation. The enzyme is widespread in soil bacteria andfungi including Pseudomonas putida (Harwood et al., J Bacteriol176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., JBacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF inPseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215(2002)), phaD in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad.Sci U S. A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (DiArch et al., Microbiol 188:117-125 (2007)), and paaJ from E. coli(Nogales et al., Microbiology 153:357-365 (2007)) also catalyze thistransformation. Several beta-ketothiolases exhibit significant andselective activities in the oxoadipyl-CoA forming direction includingbkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosaPAO1, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaDfrom P. putida. These enzymes can also be employed for the synthesis of3-oxopimeloyl-CoA, a compound structurally similar to 3-oxoadipyl-CoA.

Gene GenBank name GI# Accession # Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida pcaF 506695 AAA85138.1Pseudomonas putida pcaF 141777 AAC37148.1 Acinetobacter calcoaceticuspaaE 106636097 ABF82237.1 Pseudomonas fluorescens bkt 115360515YP_777652.1 Burkholderia ambifaria AMMD bkt 9949744 AAG06977.1Pseudomonas aeruginosa PAO1 pcaF 9946065 AAG03617.1 Pseudomonasaeruginosa PAO1

A beta-ketothiolase is also required to condense glutamyl-CoA andacetyl-CoA (FIG. 20, Step B). This transformation is not known to occurnaturally. The beta-ketothiolase candidates described above are alsoexemplary candidates for catalyzing this transformation.

2.6.1.a Aminotransferase—

Several reactions in FIGS. 20-26 are catalyzed by aminotransferases inthe EC class 2.6.1. Such enzymes reversibly transfer amino groups fromaminated donors to acceptors such as pyruvate and alpha-ketoglutarate.

Aminotransferases selective for aldehydes are required fortransaminating 2-amino-7-oxoheptanoate (FIG. 20, Step G; FIG. 21, StepQ; FIG. 26, Step M), 3-oxo-1-carboxyheptanal (FIG. 21, Step D)3-amino-7-oxoheptanoate (FIG. 21, Step Z) and 6-aminohexanal (FIG. 26,Step C; FIG. 22, Step G). An exemplary enzyme for converting aldehydesto primary amines is lysine-6-aminotransferase (EC 2.6.1.36). Thisenzyme function, converting lysine to alpha-aminoadipate semialdehyde,has been demonstrated in yeast and bacteria. Candidates from Candidautilis (Hammer et al., J Basic Microbiol 32:21-27 (1992)),Flavobacterium lutescens (Fujii et al., J Biochem. 128:391-397 (2000))and Streptomyces clavuligenus (Romero et al., Microbiol Biotechnol18:241-246 (1997)) have been characterized. A recombinantlysine-6-aminotransferase from S. clavuligenus was functionallyexpressed in E. coli (Tobin et al., J Bacteriol. 173:6223-6229 (1991)).The F. lutescens enzyme is specific to alpha-ketoglutarate as the aminoacceptor (Soda et al., Biochemistry 7:4110-4119 (1968)). Other enzymeswhich convert aldehydes to terminal amines include the dat gene productin Acinetobacter baumanii encoding 2,4-diaminobutanoate:2-ketoglutarate4-transaminase (Ikai et al., J Bacteriol. 179:5118-5125 (1997)). Inaddition to its natural substrate, 2,4-diaminobutyrate, DATtransaminates the terminal amines of lysine, 4-aminobutyrate andornithine.

GenBank Gene name GI# Accession # Organism lat 10336502 BAB13756.1Flavobacterium lutescens lat 153343 AAA26777.1 Streptomyces clavuligenusdat 6685373 P56744.1 Acinetobacter baumanii

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. The E. coli putrescine aminotransferaseis encoded by the ygjG gene and the purified enzyme was also able totransaminate cadaverine and spermidine (Samsonova et al., Microbiol 3:2(2003)). In addition, activity of this enzyme on 1,7-diaminoheptane andwith amino acceptors other than 2-oxoglutarate (e.g., pyruvate,2-oxobutanoate) has been reported (Kim et al., J Biol. Chem. 239:783-786(1964); and Samsonova et al., Microbiol 3:2 (2003)). The spuC gene ofPseudomonas aeruginosa encodes a putrescine aminotransferase with higheractivity with pyruvate as the amino acceptor than alpha-ketoglutarate(Lu et al., J Bacteriol. 184:3765-3773 (2002)).

GenBank Gene name GI# Accession # Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa

The conversion of an aldehyde to a terminal amine can also be catalyzedby gamma-aminobutyrate transaminase (GABA transaminase). This enzymenaturally interconverts succinic semialdehyde and glutamate to4-aminobutyrate and alpha-ketoglutarate and is known to have a broadsubstrate range (Liu et al., Biochemistry 43:10896-10905 (2004); andSchulz et al., Appl Environ Microbiol 56:1-6 (1990)). The two GABAtransaminases in E. coli are encoded by gabT (Bartsch et al., JBacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al., J Biol.Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus,Pseudomonas fluorescens, and Sus scrofa have been shown to react with arange of alternate substrates including 6-aminocaproic acid (Cooper,Methods Enzymol. 113:80-82 (1985)); and Scott et al., J Biol. Chem.234:932-936 (1959)).

GenBank Gene name GI# Accession # Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Enzymes that transaminate 3-oxoacids are required to convert3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate (FIG. 21, Step E),3-oxopimelate to 3-aminopimelate (FIG. 21, Step J) and3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoate (FIG. 21, Step AB).Enzymes catalyzing these exact transformations have not been identifiedto date. Beta-alanine/alpha-ketoglutarate aminotransferase (WO08027742)reacts with beta-alanine to form malonic semialdehyde, a 3-oxoacid. Thegene product of SkPYD4 in Saccharomyces kluyveri was shown topreferentially use beta-alanine as the amino group donor (Andersen etal., Gene. 124:105-109 (1993)). SkUGA1 encodes a homologue ofSaccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur.J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzymeinvolved in both beta-alanine and GABA transamination (Andersen et al.,Gene. 124:105-109 (1993)). 3-Amino-2-methylpropionate transaminasecatalyzes the transformation from methylmalonate semialdehyde to3-amino-2-methylpropionate. The enzyme has been characterized in Rattusnorvegicus and Sus scrofa and is encoded by Abat (Kakimoto et al.,Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al., MethodsEnzymol. 324:376-389 (2000)).

GenBank Gene name GI# Accession # Organism SkyPYD4 98626772 ABF58893.1Lachancea kluyveri SkUGA1 98626792 ABF58894.1 Lachancea kluyveri UGA16321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191 P50554.3Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

Several aminotransferases transaminate the amino groups of 2-oxo acidsto form amino acids. Such an enzyme is required for the transaminationof 2-oxo-7-aminoheptanoate to homolysine (FIG. 22, Step D; FIG. 26, StepM) and 2-amino-7-oxosubarate to 2,7-diaminosubarate (FIG. 26, Step K). Apromising enzyme candidate is alpha-aminoadipate aminotransferase (EC2.6.1.39), an enzyme that participates in lysine biosynthesis anddegradation in some organisms. This enzyme interconverts 2-aminoadipateand 2-oxoadipate, using alpha-ketoglutarate as the amino acceptor. Genecandidates are found in Homo sapiens (Okuno et al., Enzyme Protein47:136-148 (1993)) and Thermus thermophilus (Miyazaki et al.,Microbiology 150:2327-2334 (2004)). The Thermus thermophilus enzyme,encoded by lysN, is active with several alternate substrates includingoxaloacetate, 2-oxoisocaproate, 2-oxoisovalerate, and2-oxo-3-methylvalerate.

Gene name GI# GenBank Accession # Organism lysN 31096548 BAC76939.1Thermus thermophilus AadAT-II 46395904 Q8N5Z0.2 Homo sapiens

Another candidate is aspartate aminotransferase, an enzyme thatnaturally transfers an oxo group from oxaloacetate to glutamate, formingalpha-ketoglutarate and aspartate. Aspartate aminotransferase activityis catalyzed by, for example, the gene products of aspC from Escherichiacoli (Yagi et al., FEBS Lett. 100:81-84 (1979); and Yagi et al., MethodsEnzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi etal., J Biochem. 92:35-43 (1982)) and ASPS from Arabidopsis thaliana (dela et al., Plant J 46:414-425 (2006); Kwok et al., J Exp. Bot.55:595-604 (2004) and Wilkie et al., Protein Expr. Purif. 12:381-389(1998)). The enzyme from Rattus norvegicus has been shown totransaminate alternate substrates such as 2-aminohexanedioic acid and2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583-4589(1980)). Aminotransferases that work on other amino-acid substrates mayalso be able to catalyze this transformation. Valine aminotransferasecatalyzes the conversion of valine and pyruvate to 2-ketoisovalerate andalanine. The E. coli gene, avtA, encodes one such enzyme (Whalen et al.,J. Bacteriol. 150:739-746 (1982)). This gene product also catalyzes thetransamination of α-ketobutyrate to generate α-aminobutyrate, althoughthe amine donor in this reaction has not been identified (Whalen et al.,J. Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serCcatalyzes two reactions, phosphoserine aminotransferase andphosphohydroxythreonine aminotransferase (Lam, J. et al., Bacteriol.172:6518-6528 (1990)), and activity on non-phosphorylated substratescould not be detected (Drewke et al., FEBS. Lett. 390:179-182 (1996)).

GenBank Gene name GI# Accession # Organism aspC 16128895 NP_415448.1Escherichia coli AAT2 1703040 P23542.3 Saccharomyces cerevisiae ASP520532373 P46248.2 Arabidopsis thaliana Got2 112987 P00507 Rattusnorvegicus avtA 49176374 YP_026231.1 Escherichia coli serC 16128874NP_415427.1 Escherichia coli

2.7.2.a Phosphotransferase (Carboxy Acceptor)—

Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylicacids to phosphonic acids with concurrent hydrolysis of one ATP. StepsF, M and U in FIG. 21 require a phosphotransferase to activate thecarboxyl groups of 3-oxopimelate (Step F), 3-aminopimelate (Step M) and2-aminopimelate (Step U) to their corresponding phosphonic acids.Butyrate kinase (EC 2.7.2.7) carries out the reversible conversion ofbutyryl-phosphate to butyrate during acidogenesis in C. acetobutylicum(Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). This enzymeis encoded by either of the two buk gene products (Huang et al., J Mol.Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes arefound in C. butyricum and C. tetanomorphum (Twarog et al., J Bacteriol.86:112-117 (1963)). Related enzyme isobutyrate kinase from Thermotogamaritima has also been expressed in E. coli and crystallized (Diao etal., E. Biol. Crystallogr. 59:1100-1102 (2003); and Diao et al., JBacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes theATP-dependent phosphorylation of aspartate and participates in thesynthesis of several amino acids. The aspartokinase III enzyme in E.coli, encoded by lysC, has a broad substrate range and the catalyticresidues involved in substrate specificity have been elucidated (Keng etal., Arch. Biochem. Biophys. 335:73-81 (1996)). Two additional kinasesin E. coli are also good candidates: acetate kinase and gamma-glutamylkinase. The E. coli acetate kinase, encoded by ackA (Skarstedt et al.,J. Biol. Chem. 251:6775-6783 (1976)), phosphorylates propionate inaddition to acetate (Hesslinger et al., Mol. Microbiol 27:477-492(1998)). The E. coli gamma-glutamyl kinase, encoded by proB (Smith etal., J. Bacteriol. 157:545-551 (1984)), phosphorylates the gammacarbonic acid group of glutamate.

GenBank Gene name GI# Accession # Organism buk1 15896326 NP_349675Clostridium acetobutylicum buk2 20137415 Q97II1 Clostridiumacetobutylicum buk2 6685256 Q9X278.1 Thermotoga maritima lysC 16131850NP_418448.1 Escherichia coli ackA 16130231 NP_416799.1 Escherichia coliproB 16128228 NP_414777.1 Escherichia coli

2.8.3.a Coenzyme-A Transferase—

CoA transferases catalyze the reversible transfer of a CoA moiety fromone molecule to another. Several transformations in FIGS. 20 and 21require a CoA transferase to activate carboxylic acids to theircorresponding acyl-CoA derivatives (FIG. 20, Steps A and I; FIG. 21,Steps H, J, V). Candidate enzymes for catalyzing these transformationsinclude the gene products of cat1, cat2, and cat3 of Clostridiumkluyveri which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively(Seedorf et al., Proc. Natl. Acad. Sci U.S.A 105:2128-2133 (2008); andSohling et al., J Bacteriol. 178:871-880 (1996)). Similar CoAtransferase activities are also present in Trichomonas vaginalis (vanGrinsven et al., J Biol. Chem. 283:1411-1418 (2008)) and Trypanosomabrucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)).

GenBank Gene name GI# Accession # Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3146349050 EDK35586.1 Clostridium kluyveri TVAG_395550 123975034XP_001330176 Trichomonas vaginalis G3 Tb11.02.0290 71754875 XP_828352Trypanosoma brucei

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and3-butenoyl-CoA (Mack et al., Eur. Biochem. 226:41-51 (1994)), substratessimilar in structure to 2,3-dehydroadipyl-CoA. The genes encoding thisenzyme are gctA and gctB. This enzyme has reduced but detectableactivity with other CoA derivatives including glutaryl-CoA,2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckelet al., Eur. J Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J Biochem. 226:41-51(1994)).

GenBank Gene name GI# Accession # Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

A CoA transferase that can utilize acetyl-CoA as the CoA donor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD (beta subunit) genes (Korolev et al., Biol. Crystallogr.58:2116-2121 (2002); and Vanderwinkel et al., Biophys. Res. Commun.33:902-908 (1968)). This enzyme has a broad substrate range (Sramek etal., Arch. Biochem. Biophys. 171:14-26 (1975)) and has been shown totransfer the CoA moiety to acetate from a variety of branched and linearacyl-CoA substrates, including isobutyrate (Matthies et al., ApplEnviron. Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al.,Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel etal., Biophys. Res. Commun. 33:902-908 (1968)). This enzyme is induced atthe transcriptional level by acetoacetate, so modification of regulatorycontrol may be necessary for engineering this enzyme into a pathway(Pauli et al., Eur. J Biochem. 29:553-562 (1972)). Similar enzymes existin Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ.Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al.,Appl. Environ. Microbiol 56:1576-1583 (1990); and Wiesenborn et al.,Appl. Environ. Microbiol 55:323-329 (1989)), and Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem71:58-68 (2007)).

GenBank Gene name GI# Accession # Organism atoA 2492994 P76459.1Escherichia coli atoD 2492990 P76458.1 Escherichia coli actA 62391407YP_226809.1 Corynebacterium glutamicum cg0592 62389399 YP_224801.1Corynebacterium glutamicum ctfA 15004866 NP_149326.1 Clostridiumacetobutylicum ctfB 15004867 NP_149327.1 Clostridium acetobutylicum ctfA31075384 AAP42564.1 Clostridium saccharoperbutylacetonicum ctfB 31075385AAP42565.1 Clostridium saccharoperbutylacetonicum

The de-acylation 3-oxopimeloyl-CoA to 3-oxopimelate (FIG. 21, Step B) iscatalyzed by a 3-oxoacid-CoA transferase (EC 2.8.3.6).Succcinyl-CoA:3-oxoacid-CoA transferase, also known asbeta-ketoadipyl-CoA transferase, is encoded by pcaI and pcaJ inPseudomonas putida (Kaschabek et al., J Bacteriol. 184:207-215 (2002)).Similar enzymes based on protein sequence homology exist inAcinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)).Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are presentin Helicobacter pylori (Corthesy-Theulaz et al., J Biol. Chem.272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., ProteinExpr. Purif. 53:396-403 (2007)).

GenBank Gene name GI# Accession # Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

3.1.2.a Coa Hydrolase—

The hydrolysis of 6-aminopimeloyl-CoA to 6-aminopimelate (FIG. 20, StepI) is carried out by an acyl CoA hydrolase enzyme in the 3.1.2 family.An enzyme catalyzing this transformation has not been demonstrated todate. Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have broadsubstrate specificity and thus represent suitable candidate enzymes forhydrolyzing 6-aminopimelate. For example, the enzyme from Rattusnorvegicus brain (Robinson et al., Res. Commun. 71:959-965 (1976)) canreact with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Though itssequence has not been reported, the enzyme from the mitochondrion of thepea leaf also has a broad substrate specificity, with demonstratedactivity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA,oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant.Physiol. 94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1, from S.cerevisiae represents another candidate hydrolase (Buu et al., J. Biol.Chem. 278:17203-17209 (2003)).

GenBank Gene name GI# Accession # Organism acot12 18543355 NP_570103.1Rattus norvegicus ACH1 6319456 NP_009538 Saccharomyces cerevisiae

Another candidate hydrolase is the human dicarboxylic acid thioesterase,acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J Biol. Chem.280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J Biol.Chem. 266:11044-11050 (1991)). A similar enzyme has also beencharacterized in the rat liver (Deana et al., Biochem. Int. 26:767-773(1992)). Other potential E. coli thioester hydrolases include the geneproducts of tesA (Bonner et al., Chem. 247:3123-3133 (1972)), ybgC(Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); and (Zhuang etal., FEBS Lett. 516:161-163 (2002)), paaI (Song et al., J Biol. Chem.281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol.189:7112-7126 (2007)).

GenBank Gene name GI# Accession # Organism tesB 16128437 NP_414986Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669NP_570112 Rattus norvegicus tesA 16128478 NP_415027 Escherichia coliybgC 16128711 NP_415264 Escherichia coli paaI 16129357 NP_415914Escherichia coli ybdB 16128580 NP_415129 Escherichia coli

Yet another candidate hydrolase is the glutaconate CoA-transferase fromAcidaminococcus fermentans. This enzyme was transformed by site-directedmutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA,acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212(1997)). This suggests that the enzymes encodingsuccinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoAtransferases may also serve as candidates for this reaction step butwould require certain mutations to change their function.

GenBank Gene name GI# Accession # Organism gctA 559392 CAA57199Acidaminococcus fermentans gctB 559393 CAA57200 Acidaminococcusfermentans

4.1.1.a Carboxy-Lyase—

The decarboxylation reactions of homolysine to HMDA (FIG. 20, Step H;FIG. 21, Step S; FIG. 22, Step E; FIG. 26, Step H), 2-aminopimelate to6-ACA (FIG. 20, Step J, FIG. 21, Step AA and FIG. 26, Step E),2,7-diaminosubarate to homolysine (FIG. 26, Step L),2-amino-7-oxoheptanoate to 6-aminohexanal (FIG. 26, Step B; FIG. 22,Step F) and 2-amino-7-oxosubarate to 2-oxo-7-aminoheptanoate (FIG. 26,Step I) are catalyzed by amino acid decarboxylase enzymes. Lysinedecarboxylase (EC 4.1.1.18) catalyzes a similar transformation: thedecarboxylation of lysine to form cadaverine. Two isozymes of thisenzyme are encoded in the E. coli genome by genes cadA and ldcC. CadA isinvolved in acid resistance and is subject to positive regulation by thecadC gene product (Lemonnier et al., Microbiology 144 (Pt 3):751-760(1998)). CadC accepts hydroxylysine and S-aminoethylcysteine asalternate substrates, but 2-Aminopimelate and 6-ACA act as competitiveinhibitors to this enzyme (Sabo et al., Biochemistry 13:662-670 (1974)).Directed evolution or other enzyme engineering methods may be requiredfor this enzyme to decarboxylate 2-aminopimelate. The constitutivelyexpressed ldc gene product is less active than CadA (Lemonnier et al.,Microbiology 144 (Pt 3):751-760 (1998)). A lysine decarboxylaseanalogous to CadA was recently identified in Vibrio parahaemolyticus(Tanaka, et al., J Appl Microbiol 104:1283-1293 (2008)). The lysinedecarboxylase from Selenomonas ruminantium, encoded by ldc, bearssequence similarity to eukaryotic ornithine decarboxylases, and acceptsboth L-lysine and L-ornithine as substrates (Takatsuka et al., Biosci.Biotechnol Biochem. 63:1843-1846 (1999)). Active site residues wereidentified and engineered to alter the substrate specificity of theenzyme (Takatsuka et al., J Bacteriol. 182:6732-6741 (2000)).

GenBank Gene name GI# Accession # Organism cadA 145458 AAA23536.1Escherichia coli ldcC 1786384 AAC73297.1 Escherichia coli ldc 13124043O50657.1 Selenomonas ruminantium cadA 44886078 AB124819.1 Vibrioparahaemolyticus

Several ornithine decarboxylase enzymes (EC 4.1.1.17) exhibit activityon lysine and other similar compounds. Such enzymes are found inNicotiana glutinosa (Lee et al., Biochem. J 360:657-665 (2001)),Lactobacillus sp. 30a (Guirard et al., J Biol. Chem. 255:5960-5964(1980)) and Vibrio vulnificus (Lee et al., J Biol. Chem. 282:27115-27125(2007)). The enzymes from Lactobacillus sp. 30a (Momany et al., J Mol.Biol. 252:643-655 (1995)) and V. vulnificus have been crystallized. TheV. vulnificus enzyme efficiently catalyzes lysine decarboxylation andthe residues involved in substrate specificity have been elucidated (Leeet al., J Biol. Chem. 282:27115-27125 (2007)). A similar enzyme has beencharacterized in Trichomonas vaginalis but the gene encoding this enzymeis not known (Yarlett et al., Biochem. J 293 (Pt 2):487-493 (1993)).

GenBank Gene name GI# Accession # Organism AF323910.1: 1 . . . 129912007488 AAG45222.1 Nicotiana glutinosa odc1 1169251 P43099.2Lactobacillus sp. 30a VV2_1235 27367615 NP_763142.1 Vibrio vulnificus

Keto-acid decarboxylase enzymes are required to convert2-oxo-7-aminoheptanoate to 6-aminohexanal (Step F of FIG. 22; Step G ofFIG. 26) and 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate (Step A ofFIG. 26). The decarboxylation of keto-acids is catalyzed by a variety ofenzymes with varied substrate specificities, including pyruvatedecarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7),alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoaciddecarboxylase. Pyruvate decarboxylase (PDC), also termed keto-aciddecarboxylase, is a key enzyme in alcoholic fermentation, catalyzing thedecarboxylation of pyruvate to acetaldehyde. The enzyme fromSaccharomyces cerevisiae has a broad substrate range for aliphatic2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvateand 2-phenylpyruvate (Henning et al., Appl. Environ. Microbiol.72:7510-7517 (2006)). This enzyme has been extensively studied,engineered for altered activity, and functionally expressed in E. coli(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001); Li, H.and F. Jordan, Biochemistry. 38:10004-10012 (1999); and ter Schure etal., Appl. Environ. Microbiol. 64:1303-1307 (1998)). The PDC fromZymomonas mobilus, encoded by pdc, also has a broad substrate range andhas been a subject of directed engineering studies to alter the affinityfor different substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The crystal structure of this enzyme is available(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Otherwell-characterized PDC candidates include the enzymes from Acetobacterpasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) andKluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263(2002)).

GenBank Gene name GI# Accession # Organism pdc 118391 P06672.1 Zymomonasmobilus pdc1 30923172 P06169 Saccharomyces cerevisiae pdc 20385191AM21208 Acetobacter pasteurians pdc1 52788279 Q12629 Kluyveromyceslactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.,Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry42:1820-1830 (2003)). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity(K_(m)) of naturally and non-naturally occurring substrates (Siegert etal., Protein Eng Des Sel 18:345-357 (2005)). The properties of thisenzyme have been further modified by directed engineering (Lingen etal., Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem.4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa, encoded bymdlC, has also been characterized experimentally (Barrowman et al., FEMSMicrobiology Letters 34:57-60 (1986)). Additional gene candidates fromPseudomonas stutzeri, Pseudomonas fluorescens and other organisms can beinferred by sequence homology or identified using a growth selectionsystem developed in Pseudomonas putida (Henning et al., Appl. Environ.Microbiol. 72:7510-7517 (2006)).

GenBank Gene name GI# Accession # Organism mdlC 3915757 P20906.2Pseudomonas putida mdlC 81539678 Q9HUR2.1 Pseudomonas aeruginosa dpgB126202187 ABN80423.1 Pseudomonas stutzeri ilvB-1 70730840 YP_260581.1Pseudomonas fluorescens

A third enzyme capable of decarboxylating 2-oxoacids isalpha-ketoglutarate decarboxylase (KGD). The substrate range of thisclass of enzymes has not been studied to date. The KDC fromMycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US.A102:10670-10675 (2005)) has been cloned and functionally expressed inother internal projects at Genomatica. However, it is not an idealcandidate for strain engineering because it is large (˜130 kD) andGC-rich. KDC enzyme activity has been detected in several species ofrhizobia including Bradyrhizobium japonicum and Mesorhizobium loti(Green et al., J Bacteriol. 182:2838-2844 (2000)). Although theKDC-encoding gene(s) have not been isolated in these organisms, thegenome sequences are available and several genes in each genome areannotated as putative KDCs. A KDC from Euglena gracilis has also beencharacterized but the gene associated with this activity has not beenidentified to date (Shigeoka and Nakano, Arch. Biochem. Biophys.288:22-28 (1991)). The first twenty amino acids starting from theN-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 1) (Shigeokaand Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene could beidentified by testing candidate genes containing this N-terminalsequence for KDC activity.

GenBank Gene name GI# Accession # Organism kgd 160395583 O50463.4Mycobacterium tuberculosis kgd 27375563 NP_767092.1 Bradyrhizobiumjaponicum kgd 13473636 NP_105204.1 Mesorhizobium loti

A fourth candidate enzyme for catalyzing this reaction is branched chainalpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shownto act on a variety of compounds varying in chain length from 3 to 6carbons (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988); and Smitet al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme inLactococcus lactis has been characterized on a variety of branched andlinear substrates including 2-oxobutanoate, 2-oxohexanoate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate andisocaproate isocaproate (Smit et al., Appl Environ Microbiol. 71:303-311(2005)). The enzyme has been structurally characterized (Berg et al.,Science. 318:1782-1786 (2007)). Sequence alignments between theLactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonasmobilus indicate that the catalytic and substrate recognition residuesare nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357(2005)), so this enzyme would be a promising candidate for directedengineering. Decarboxylation of alpha-ketoglutarate by a BCKA wasdetected in Bacillus subtilis; however, this activity was low (5%)relative to activity on other branched-chain substrates (Oku and Kaneda,J Biol Chem. 263:18386-18396 (1988)) and the gene encoding this enzymehas not been identified to date. Additional BCKA gene candidates can beidentified by homology to the Lactococcus lactis protein sequence. Manyof the high-scoring BLASTp hits to this enzyme are annotated asindolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvatedecarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation ofindolepyruvate to indoleacetaldehyde in plants and plant bacteria.

GenBank Gene name GI# Accession # Organism kdcA 44921617 AAS49166.1Lactococcus lactis

Recombinant branched chain alpha-keto acid decarboxylase enzymes derivedfrom the E1 subunits of the mitochondrial branched-chain keto aciddehydrogenase complex from Homo sapiens and Bos taurus have been clonedand functionally expressed in E. coli (Davie et al., J. Biol. Chem.267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). Inthese studies, the authors found that co-expression of chaperonins GroELand GroES enhanced the specific activity of the decarboxylase by500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). Theseenzymes are composed of two alpha and two beta subunits.

GenBank Gene name GI# Accession # Organism BCKDHB 34101272 NP_898871.1Homo sapiens BCKDHA 11386135 NP_000700.1 Homo sapiens BCKDHB 115502434P21839 Bos taurus BCKDHA 129030 P11178 Bos taurus

4.1.2.a

The condensation of pyruvate with 4-aminobutanal (FIG. 22, Step A) orglutamate-5-semialdehyde (FIG. 27, Step A) is catalyzed by an aldehydelyase in the EC class 4.1.2. A variety of aldehyde lyase enzymes utilizepyruvate as an acceptor; however, none have been demonstrated to utilize4-aminobutanal or glutamate-5-semialdehyde as a donor. The enzyme4-hydroxy-2-oxopimelate (HODH) aldolase (EC 4.1.2.-), condenses succinicsemialdehyde and pyruvate to catalyze the formation of4-hydroxy-2-oxopimelate. This enzyme is a divalent metal ion-dependentclass II aldolase, catalyzing the final step of 4-hydroxyphenylaceticacid degradation in E. coli C, E. coli W, and other organisms. In thenative context, the enzyme functions in the degradative direction. Thereverse (condensation) reaction is thermodynamically unfavorable;however the equilibrium can be shifted through coupling HODH aldolasewith downstream pathway enzymes that work efficiently on reactionproducts. Such strategies have been effective for shifting theequilibrium of other aldolases in the condensation direction (Nagata etal., Appl Microbiol Biotechnol 44:432-438 (1995); and Pollard et al.,Appl Environ. Microbiol 64:4093-4094 (1998)). The E. coli C enzyme,encoded by hpcH, is able to condense a range of aldehyde acceptors withpyruvate and has recently been crystallized (Rea et al., J Mol. Biol.373:866-876 (2007); and

Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme isencoded by hpaI (Prieto et al., J Bacteriol. 178:111-120 (1996)).

GenBank Gene name GI# Accession # Organism hpcH 633197 CAA87759.1Escherichia coli C hpaI 38112625 AAR11360.1 Escherichia coli W

Another pyruvate-utilizing aldehyde lyase is 2-dehydro-3-deoxyglucaratealdolase (DDGA, EC 4.1.2.20), a type II aldolase that participates inthe catabolic pathway for D-glucarate/galactarate utilization in E.coli. The natural donor of this enzyme is tartronate semialdehyde, butthis enzyme has a broad substrate specificity and has been shown toreversibly condense a wide range of aldehydes with pyruvate (Fish etal., Methods Enzymol. 9:529-534 (1966)). The crystal structure of thisenzyme has been determined and a catalytic mechanism proposed (Izard etal., EMBO J 19:3849-3856 (2000)). Additional candidate DDGA enzymes arefound in Leptospira interrogans (118) and Sulfolobus solfataricus(Buchanan et al., Biochem. J 343 Pt 3:563-570 (1999)). The S.solfataricus enzyme is highly thermostable and was cloned and expressedin E. coli (Buchanan et al., Biochem. J 343 Pt 3:563-570 (1999)).

GenBank Gene name GI# Accession # Organism garL 1176153 P23522.2Escherichia coli LA_1624 24195249 AAN48823.1 Leptospira interrogansAJ224174.1:1..885 2879782 CAA11866.1 Sulfolobus solfataricus

4.2.1.a Hydro-Lyase—

Two reactions in FIGS. 20 and 22 employ enzymes in the dehydratase class(EC 4.1.2). The dehydration of 3-hydroxy-6-aminopimeloyl-CoA (FIG. 20,Step D) is catalyzed by an enoyl-CoA hydratase. This reaction is notknown to occur naturally; however the ability to dehydrate3-hydroxyacyl-CoA derivatives is widespread. Enoyl-CoA hydratases (EC4.2.1.17) catalyze the dehydration of a range of 3-hydroxyacyl-CoAsubstrates (Agnihotri et al., Bioorg. Med. Chem. 11:9-20 (2003; Conradet al., J Bacteriol. 118:103-111 (1974) and Roberts et al., Arch.Microbiol 117:99-108 (1978)). The enoyl-CoA hydratase of Pseudomonasputida, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoAto crotonyl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978)).Additional enoyl-CoA hydratase candidates are phaA and phaB, of P.putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc.Natl. Acad. Sci U.S.A 95:6419-6424 (1998)). The gene product of pimF inRhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratasethat participates in pimeloyl-CoA degradation (Harrison et al.,Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coligenes have been shown to demonstrate enoyl-CoA hydratase functionalityincluding maoC (Park et al., J Bacteriol. 185:5391-5397. 2003), paaF(Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl.Biochem. Biotechnol 113-116:335-346 (2004) and (Park et al., BiotechnolBioeng 86:681-686 (2004)) and paaG (Park et al., J Bacteriol.185:5391-5397. 2003), paaF (Ismail et al., Eur. J Biochem. 270:3047-3054(2003); Park et al., Appl. Biochem. Biotechnol 113-116:335-346 (2004)and (Park et al., Biotechnol Bioeng 86:681-686 (2004)).

Gene GenBank name GI# Accession # Organism ech 26990073 NP_745498.1Pseudomonas putida paaA 26990002 NP_745427.1 Pseudomonas putida paaB26990001 NP_745426.1 Pseudomonas putida phaA 106636093 ABF82233.1Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonasfluorescens pimF 39650635 CAE29158 Rhodopseudomonas palustris maoC16129348 NP_415905.1 Escherichia coli paaF 16129354 NP_415911.1Escherichia coli paaG 16129355 NP_415912.1 Escherichia coli

3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase,is an enoyl-CoA hydratase that dehydrates 3-hydroxyisobutyryl-CoA toform crotonyl-CoA. Crotonase enzymes are required for n-butanolformation in some organisms, particularly Clostridial species, and alsocomprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle inthermoacidophilic Archaea of the genera Sulfolobus, Acidianus, andMetallosphaera. Exemplary genes encoding crotonase enzymes can be foundin C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311 (2008); andBoynton et al., J Bacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmeret al., FEBS Lett. 21:351-354. 1972)), and Metallosphaera sedula (Berget al., Science. 318:1782-1786 (2007)) though the sequence of the lattergene is not known.

Gene GenBank name GI# Accession # Organism crt 15895969 NP_349318.1Clostridium acetobutylicum crt1 153953091 YP_001393856.1 Clostridiumkluyveri

Alternatively, the E. coli gene products of fadA and fadB encode amultienzyme complex involved in fatty acid oxidation that exhibitsenoyl-CoA hydratase activity (Nakahigashi et al., Nucleic Acids Res.18:4937 (1990); Yang et al., J Bacteriol. 173:7405-7406 (1991) and Yanget al., Biochemistry 30:6788-6795 (1991)). Knocking out a negativeregulator encoded by fadR can be utilized to activate the fadB geneproduct (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI andfadJ genes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805(2003)).

Gene name GI# GenBank Accession # Organism fadA 49176430 YP_026272.1Escherichia coli fadB 16131692 NP_418288.1 Escherichia coli fadI16130275 NP_416844.1 Escherichia coli fadJ 16130274 NP_416843.1Escherichia coli fadR 16129150 NP_415705.1 Escherichia coli

2-Oxo-7-aminohept-3-enoate is formed from the dehydration of2-oxo-4-hydroxy-7-aminoheptanoate (FIG. 22, Step B). The dehydration of2-amino-5-hydroxy-7-oxosubarate to 2-amino-5-ene-7-oxosubarate (FIG. 27,Step B) is a similar transformation. Enzymes catalyzing these exactreactions are not known to occur naturally. A candidate enzyme thatcatalyzes a similar reaction is OHED hydratase, which naturallydehydrates 2-oxo-4-hydroxy-hepta-1,7-dioate (HODH) to2-oxo-hept-4-ene-1,7-dioate (OHED) HODH is similar in structure to thedesired substrates. This enzyme requires magnesium as a cofactor (Burkset al., J. Am. Chem. Soc. 120 (1998)). OHED hydratase enzyme candidateshave been identified and characterized in E. coli C (Izumi et al., JMol. Biol. 370:899-911 (2007; and Roper et al., Gene 156:47-51 (1995))and E. coli W (Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequencecomparison reveals homologs in a range of bacteria, plants and animals.Enzymes with highly similar sequences are contained in Klebsiellapneumonia (91% identity, evalue=2e-138) and Salmonella enterica (91%identity, evalue=4e-138), among others.

Gene name GI# GenBank Accession # Organism hpcG 556840 CAA57202.1Escherichia coli C hpaH 757830 CAA86044.1 Escherichia coli W hpaH150958100 ABR80130.1 Klebsiella pneumoniae Sari_01896 160865156ABX21779.1 Salmonella enterica

An alternate enzyme candidate for catalyzing this reaction is fumarase,also known as fumarate hydratase (EC 4.2.1.2). E. coli has threefumarases: FumA, FumB, and FumC that are regulated by growth conditions.FumB is oxygen sensitive and only active under anaerobic conditions.FumA is active under microanaerobic conditions, and FumC is the onlyactive enzyme in aerobic growth (Guest et al., J Gen Microbiol.131:2971-2984 (1985); Tseng et al., J Bacteriol 183:461-467 (2001) andWoods et al., Biochim Biophys Acta 954:14-26 (1988)). FumC has beenshown to dehydrate alternate substrates including tartrate andthreo-hydroxyaspartate (Teipel et al., J Biol. Chem. 243:5684-5694(1968)). A wealth of structural information is available for FumC andresearchers have successfully engineered the enzyme to alter activity,inhibition and localization (Weaver et al., D Biol Crystallogr.61:1395-1401 (2005)). Additional fumarate hydratase enzymes are found inEscherichia coli (Estevez et al., Protein Sci 11:1552-1557 (2002); Hong,et al., Biotechnol. Bioprocess Eng. 9:252-255 (2005)) and Rose et al.,Proc Natl Acad Sci US. A 101:3393-3397 (2004)), Corynebacteriumglutamicum (Genda et al., Biotechnol Biochem. 70:1102-1109 (2006)),Campylobacter jejuni (Smith et al., Cell Biol 31:961-975 (1999)),Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55(1998)), and Rattus norvegicus (Kobayashi et al., J Biochem.89:1923-1931 (1981)). The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

Gene GenBank name GI# Accession # Organism fumA 81175318 P0AC33Escherichia coli K12 fumB 33112655 P14407 Escherichia coli K12 fumC120601 P05042.1 Escherichia coli K12 fumC 39931596 Q8NRN8.1Corynebacterium glutamicum fumC 9789756 O69294.1 Campylobacter jejunifumC 75427690 P84127 Thermus thermophilus fumH 120605 P14408.1 Rattusnorvegicus MmcB 147677691 YP_001211906 Pelotomaculum thermopropionicumMmcC 147677692 YP_001211907 Pelotomaculum thermopropionicum

Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), anenzyme that naturally dehydrates 2-methylmalate to mesaconate. Thisenzyme has been studied in Methanocaldococcus jannaschii in the contextof the pyruvate pathway to 2-oxobutanoate, where it has been shown tohave a broad substrate specificity (Drevland et al., J Bacteriol.189:4391-4400 (2007)). This enzyme activity was also detected inClostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticuswhere it is thought to participate in glutamate degradation (Kato etal., Arch. Microbiol 168:457-463 1997)). The M. jannaschii proteinsequence does not bear significant homology to genes in these organisms.

Gene GenBank name GI# Accession # Organism leuD 3122345 Q58673.1Methanocaldococcus jannaschii

5.4.3.a Aminomutase—Several reactions in FIG. 21 involve shifting asecondary amine from the 3- to the 2-position (FIG. 21, Steps P, R, T).A promising enzyme candidate for catalyzing these transformations islysine 2,3-aminomutase (EC 5.4.3.2), an enzyme that naturally convertslysine to (3S)-3,6-diaminohexanoate, reversibly shifting an amine groupfrom the 2- to the 3-position. The enzyme is found in bacteria thatferment lysine to acetate and butyrate, including Fusobacteriumnucleatum (kamA) (Barker et al., J Bacteriol. 152:201-207 (1982)) andClostridium subterminale (kamA) (Chirpich et al., J Biol. Chem.245:1778-1789 (1970)). The enzyme from Clostridium subterminale has beencrystallized (117). An enzyme encoding this function is also encoded byyodO in Bacillus subtilis (Chen et al., Biochem. J. 348 Pt 3:539-549(2000)). The enzyme utilizes pyridoxal 5′-phosphate as a cofactor,requires activation by S-adenosylmethoionine, and is stereoselective forL-lysine. The enzyme has not been shown to react with alternatesubstrates, so directed evolution or other engineering methods may berequired for this enzyme to react with the non-natural substrates3-amino-7-oxohexanoate, 3,7-diaminoheptanoate and 3-aminopimelate. Forexample, Cargill has developed a novel 2,3-aminomutase enzyme derivedfrom lysine-2,3-aminomutase that converts L-alanine to β-alanine (Liaoet al., United States Patent 20050221466 (2005)).

GenBank Gene name GI# Accession # Organism kamA 75423266 Q9XBQ8.1Clostridium subterminale kamA 81485301 Q8RHX4 Fusobacterium nucleatumyodO 4033499 O34676.1 Bacillus subtilis

Other enzymes with 2,3-aminomutase activity include tyrosine2,3-aminomutase (EC 5.4.3.6) and leucine 2,3-aminomutase (EC 5.4.3.7).Tyrosine 2,3-aminomutase participates in tyrosine biosynthesis,reversibly converting tyrosine to3-amino-3-(4-hydroxyphenyl)-propionoate by shifting an amine from the 2-to the 3-position. In Streptomyces globisporus the enzyme has also beenshown to react with tyrosine derivatives (Christenson et al.,Biochemistry 42:12708-12718 (2003)); however, the sequence of thisenzyme is not yet available. Leucine 2,3-aminomutase converts L-leucineto beta-leucine during leucine biosynthesis and degradation. A leucine2,3-aminomutase-specific assay detected enzyme activity in manyorganisms (Poston et al., Methods Enzymol. 166:130-135 (1988)) but genesencoding the enzyme have not been identified to date.

6.2.1.a Acid-Thiol Ligase—

The activation of carboxylic acids to acyl-CoA derivatives is catalyzedby CoA acid-thiol ligases or CoA synthetases in the EC class 6.2.1 (theterms ligase, synthetase, and synthase are used herein interchangeablyand refer to the same enzyme class). Such enzymes couple the energeticcost of thioester bond formation to the hydrolysis of ATP into ADP orAMP. Several ADP-forming CoA ligases have been demonstrated to react inthe reverse direction, removing the CoA moiety from acyl-CoA moleculesand concomitantly forming ATP. Reversible CoA ligases are required tode-acylate 6-aminopimeloyl-CoA (FIG. 20, Step I) and 3-oxopimeloyl-CoA(FIG. 21, Step B), whereas AMP or ADP forming ligases can acylate3-oxopimelate (FIG. 21, Step H), 3-aminopimelate (FIG. 21, Step K) and2-aminopimelate (FIG. 21, Step V). Enzymes catalyzing these exacttransformations have not been characterized to date; however, severalenzymes with broad substrate specificities have been described in theliterature.

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme thatcouples the conversion of acyl-CoA esters to their corresponding acidswith the concomitant synthesis of ATP. ACD I from Archaeoglobusfulgidus, encoded by AF1211, was shown to operate on a variety of linearand branched-chain substrates including isobutyrate, isopentanoate, andfumarate (Musfeldt et al., J Bacteriol. 184:636-644 (2002)). A secondreversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was alsoshown to have a broad substrate range with high activity on cycliccompounds phenylacetate and indoleacetate (Musfeldt et al., J Bacteriol.184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotatedas a succinyl-CoA synthetase) accepts propionate, butyrate, andbranched-chain acids (isovalerate and isobutyrate) as substrates, andwas shown to operate in the forward and reverse directions (Brasen etal., Arch. Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed thebroadest substrate range of all characterized ACDs, reacting withacetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA(Brasen et al., Arch. Microbiol 182:277-287 (2004)). Directed evolutionor engineering can be used to modify this enzyme to operate at thephysiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen et al.,Arch. Microbiol 182:277-287 (2004); and Musfeldt et al., J Bacteriol.184:636-644 (2002)). An additional candidate is the enzyme encoded bysucCD in E. coli, which naturally catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Buck et al., Biochemistry24:6245-6252 (1985)).

GenBank Gene name GI# Accession # Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 AF1983 11499565 NP_070807.1Archaeoglobus fulgidus DSM 4304 scs 55377722 YP_135572.1 Haloarculamarismortui PAE3250 18313937 NP_560604.1 Pyrobaculum aerophilum str. IM2sucC 16128703 NP_415256.1 Escherichia coli sucD 1786949 AAC73823.1Escherichia coli

Another candidate enzyme is the AMP-forming pimeloyl-CoA ligase (EC6.2.1.14) which naturally activates pimelate to pimeloyl-CoA duringbiotin biosynthesis in gram-positive bacteria. The enzyme fromPseudomonas mendocina, cloned into E. coli, was shown to accept thealternate substrates hexanedioate and nonanedioate (Binieda et al.,Biochem. J 340 (Pt 3):793-801 (1999)). Other pimeloyl-CoA ligasecandidates are found in Bacillus subtilis (Bower et al., J Bacteriol.178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillussphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690 (1992)).

GenBank Gene name GI# Accession # Organism pauA 15596214 NP_249708.1Pseudomonas mendocina bioW 50812281 NP_390902.2 Bacillus subtilis bioW115012 P22822.1 Lysinibacillus sphaericus

Additional CoA-ligases include the rat dicarboxylate-CoA ligase forwhich the sequence is yet uncharacterized (Vamecq et al., Biochem J230:683-693 (1985)), either of the two characterized phenylacetate-CoAligases from P. chrysogenum (Lamas-et al., Maceiras, J 395:147-155(2006); and Wang et al., Biophys. Res. Commun. 360:453-458 (2007)) andthe phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco etal., J Biol. Chem. 265:7084-7090 (1990)). Acetoacetyl-CoA synthetasesfrom Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419(2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994(2003)) naturally catalyze the ATP-dependant conversion of acetoacetateinto acetoacetyl-CoA.

Gene name GI# GenBank Accession # Organism phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida AACS 21313520NP_084486.1 Mus musculus AACS 31982927 NP_076417.2 Homo sapiens

Example XXVII Additional Pathways for Production of Hexamethylenediaminefrom 6-Aminocaproate

FIG. 24 provides additional pathways for HMDA production and is furtherto FIG. 13 and Example XX above. Arrows for Steps 0 and P indicate thedirect conversion of 6-aminocaproate and 6-acetamidohexanoate to6-aminocaproic semialdehyde and 6-acetamidohexanal, respectively. Thesereactions are catalyzed by a reductase in EC class 1.2.1.e. For adescription of enzyme candidates, see Example XXVI (EC 1.2.1.e).

Example XXVIII Pathways for Production of 6-Aminocaproate from Adipate

FIG. 25 provides additional pathways for 6-ACA production and is furtherto FIG. 10 and Example XVI above. The conversion of adipate to adipatesemialdehyde (FIG. 25, Step X) is catalyzed by an enzyme with adipatereductase functionality. Adipate kinase catalyzes the formation ofadipylphosphate from adipate (FIG. 25, Step Y). Adipate semialdehyde isformed from adipylphosphate by adipylphosphate reductase (FIG. 25, StepZ). Enzyme candidates for catalyzing these transformations are describedin Example XXVI.

Example XXIX Pathway for Production of Levulinic Acid

Levulinic acid (LA), also known as 4-oxopentanoic acid and 4-ketovalericacid, is a precursor to nylon-like polymers, synthetic rubbers andplastics. It is also a precursor of other commodity chemicals such asmethyltetrahydrofuran, valerolactone and ethyl levulinate. Otherpotential applications include use as a fuel extender and abiodegradable herbicide/pesticide. It is traditionally prepared bytreating cellulosic biomass with strong acids such as hydrochloric andsulfuric acids. This process has the disadvantages of low LA yield andnumerous byproducts. More recently, the Biofine Process was developedwhich converts cellulosic biomass into LA, formic acid and furfural at ayield of 70% the theoretical maximum (Hayes et al., “The biofineprocess—production of levulinic acid, furfural and formic acid fromlignocellulosic feedstock” p. 139-164. In Biorefineries: IndustrialProcesses and Products. Wiley, Weinheim, Germany (2006)). Describedherein is a process for selectively producing LA from sugar or syngasfeedstocks using a microbial organism.

The maximum theoretical yield of LA from glucose is 1.45 moles of LA permole glucose utilized (0.938 g/g), according to the following equation:

Glucose(C₆H₁₂O₂)+1.27CO₂→1.45LA(C₅H₈O₃)+0.18H₂O

LA is produced from the central metabolites succinyl-CoA and acetyl-CoAin three enzymatic steps. In the first step, acetyl-CoA and succinyl-CoAare condensed by a beta-ketothiolase to form 3-oxoadipyl-CoA (Step A ofFIG. 25). The CoA moiety is subsequently removed by a CoA hydrolase,transferase or ligase (Steps E/F/G of FIG. 25). In the final step of thepathway, 3-oxoadipate is decarboxylated to LA (Step AA of FIG. 25).

The decarboxylation of 3-oxoadipate to LA can occur enzymatically orspontaneously. In E. coli, several 3-oxoacids produced during amino acidbiosynthesis have been shown to undergo spontaneous decarboxylation(Boylan et al., Biochem. Biophys. Res Commun. 85:190-197 (1978)). Anenzyme catalyzing the decarboxylation of 3-oxoadipate to LA has not beendemonstrated to our knowledge. An exemplary enzyme candidate catalyzinga similar reaction is acetoacetate decarboxylase (EC 4.1.1.4). Theacetoacetate decarboxylase from Clostridium acetobutylicum, encoded byadc, has a broad substrate specificity and has been shown todecarboxylate 3-oxopentanoate, 2-oxo-3-phenylpropionic acid and2-methyl-3-oxobutyrate (Benner et al., J. Am. Chem. Soc. 103:993-994(1981) and Rozzel et al., J. Am. Chem. Soc. 106:4937-4941 (1984)). Anacetoacetate decarboylase has also been characterized in Clostridiumbeijerinckii (Ravagnani et al., Mol. Microbiol 37:1172-1185 (2000)). Theacetoacetate decarboxylase from Bacillus polymyxa, characterized incell-free extracts, also has a broad substrate specificity for 3-ketoacids and can decarboxylate 3-oxopentanoate (Matiasek et al., Curr.Microbiol 42:276-281 (2001)). The gene encoding this enzyme has not beenidentified to date and the genome sequence of B. polymyxa is not yetavailable. Another adc is found in Clostridiumsaccharoperbutylacetonicum (Kosaka, et al., Biosci. Biotechnol Biochem.71:58-68 (2007)).

GenBank Gene name GI# Accession # Organism adc 15004868 NP_149328.1Clostridium acetobutylicum adc 31075386 AAP42566.1 Clostridiumsaccharoper- butylacetonicum cbei_3835 150018652 YP_001310906.1Clostridium beijerinckii

Example XXX In Silico Knockout Strategies for Production of Adipate,6-ACA and HMDA

This example describes gene disruption strategies for production ofadipate 6-aminocaproic acid (6-ACA) and hexamethylenediamine (HMDA).

Described below in more detail are sets of enzymatic activities that canbe reduced by appropriate gene disruptions or deletions in a productionhost engineered to contain the adipate, 6-aminocaproic acid (6-ACA) andhexamethylenediamine (HMDA) production pathways, for example, pathwaysusing succinyl CoA and acetyl CoA as precursors.

OptKnock is a bilevel computational framework formulated with theoverall objective of developing genetically stable overproducingmicroorganisms. Specifically, the framework examines the completenetwork of a microorganism in order to suggest genetic manipulationsthat force the desired biochemical to become an obligatory byproduct ofcell growth. By coupling biochemical production with cell growth throughstrategically placed gene disruptions or deletions, the growth selectionpressures imposed on the engineered strains after long periods of timein a bioreactor lead to improvements in performance as a result of thecompulsory growth-coupled biochemical production. Lastly, in the case ofa gene deletion, there is negligible possibility of the designed strainsreverting to their wild-type states because the genes selected byOptKnock are completely removed from the genome.

Growth-coupled biochemical production can be visualized in the contextof the biochemical production envelopes of a typical metabolic networkcalculated using an in silico model. These limits are obtained by fixingthe uptake rate(s) of the limiting substrate(s) to their experimentallymeasured value(s) and calculating the maximum and minimum rates ofbiochemical production at each attainable level of growth. Althoughexceptions exist, typically the production of a desired biochemical isin direct competition with biomass formation for intracellularresources. Thus, enhanced rates of biochemical production will generallyresult in sub-maximal growth rates. The knockouts suggested by OptKnockare designed to restrict the allowable solution boundaries forcing achange in metabolic behavior from the wild-type strain. Although theactual solution boundaries for a given strain will expand or contract asthe substrate uptake rate(s) increase or decrease, each experimentalpoint should lie within its calculated solution boundary. Plots such asthese allow visualization of how close strains are to their performancelimits or, in other words, how much room is available for improvement.The OptKnock framework has been used to identify promising gene deletionstrategies for biochemical overproduction and establishes a systematicframework that will naturally encompass future improvements in metabolicand regulatory modeling frameworks.

Described below are sets of enzyme activities that should be absent,attenuated, or eliminated for creating host organisms that achievegrowth-coupled adipate, 6-ACA or HMDA production upon the addition ofthe biosynthetic pathway that proceeds through succinyl-CoA andacetyl-CoA. To enumerate all potential strategies, an optimizationtechnique, termed integer cuts, has been implemented which entailsiteratively solving the OptKnock problem with the incorporation of anadditional constraint referred to as an integer cut at each iteration.

The OptKnock algorithm was used to identify designs based on astoichiometric model of Escherichia coli metabolism. Assumptions include(i) a glucose uptake rate of 10 mmol/gdw/hr; (ii) anaerobic ormicroaerobic conditions; and (iii) a minimum non-growth associatedmaintenance requirement of 4 mmol/gdw/hr. Table 12 provides a list ofall the reaction stoichiometries and the associated genes known to beassociated with the reactions identified for deletion in the strategies.Table 13 provides a list of the metabolite abbreviations, thecorresponding names and locations of all the metabolites thatparticipate in the reactions listed in Table 12. The growth-coupledproductions designs for adipic acid, GACA and HMDA are provided inTables 14-16. The product formation rates shown in Tables 14-16 are inmmol/gDCW·hr. The basis glucose uptake rate is 10 mmol/gDCW·hr and thebiomass formation rate is shown in units of 1/hr. These tables list thereactions that are knocked out in a particular strategy, the anticipatedproduct and biomass yields. Although the designs were identified using ametabolic model of E. coli metabolism, and the gene names listed arespecific to E. coli, the method of choosing the metabolic engineeringstrategies and also the designs themselves are applicable to any HMDA,6-ACA or adipate-producing organism. Thus the designs are essentiallylists of enzymatic transformations whose activity is to be eithereliminated, attenuated, or initially absent from a microorganism toprovide the growth coupled production of adipate, GACA and HMDA.

The key criterion for prioritizing the final selection of designs wasthe growth-coupled yield of each of the products. To examine this,production cones were constructed for each strategy by first maximizingand, subsequently minimizing the product yields at different rates ofbiomass formation, as described above. If the rightmost boundary of allpossible phenotypes of the mutant network is a single point, it impliesthat there is a unique optimum yield of the product at the maximumbiomass formation rate possible in the network. In other cases, therightmost boundary of the feasible phenotypes is a vertical line,indicating that at the point of maximum biomass the network can make anyamount of the product in the calculated range, including the lowestamount at the bottommost point of the vertical line. Such designs weregiven a lower priority.

The metabolic engineering strategies described below assume that theorganism can produce adipate, 6-ACA or HMDA via the succinyl CoA andacetyl-CoA utilizing pathway. The construction of a recombinant hostorganism capable of producing these products via the pathway isdescribed herein.

Strain Construction:

In order to validate the computational predictions proposed in thisreport, the strains are constructed, evolved, and tested. Escherichiacoli K-12 MG1655 housing the succinyl-CoA-acetyl-CoA pathway serves asthe strain into which the deletions are introduced. The strains areconstructed by incorporating in-frame deletions using homologousrecombination via the λ Red recombinase system of Datsenko and Wanner(Proc. Natl. Acad. Sci. USA 97(12):6640-6645 2000)). The approachinvolves replacing a chromosomal sequence, that is, the gene targetedfor removal, with a selectable antibiotic resistance gene, which itselfis later removed. The knockouts are integrated one by one into therecipient strain. No antibiotic resistance markers remain after eachdeletion, allowing accumulation of multiple mutations in each targetstrain. The deletion technology completely removes the gene targeted forremoval so as to substantially reduce the possibility of the constructedmutants reverting back to the wild-type.

Shake Flask Characterization:

As intermediate strains are being constructed, strain performance isquantified by performing shake flask fermentations. Anaerobic conditionsare obtained by sealing the flasks with a rubber septum and thensparging the medium with nitrogen. For strains where growth is notobserved under strict anaerobic conditions, microaerobic conditions canbe applied by covering the flask with foil and poking a small hole forlimited aeration. All experiments are performed using M9 minimal mediumsupplemented with glucose unless otherwise stated. Pre-cultures aregrown overnight and used as inoculum for a fresh batch culture for whichmeasurements are taken during exponential growth. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm), and the glucose uptake rate by monitoring carbon source depletionover time. The products, ethanol, and organic acids are analyzed byGC-MS or HPLC using routine procedures. Triplicate cultures are grownfor each strain.

Batch Fermenter Testing:

The performance of select strains is tested in anaerobic, pH-controlledbatch fermentations. This allows reliable quantification of the growth,glucose uptake, and formation rates of all products, as well as ensurethat the accumulation of acidic fermentation products will not limitcell growth. In addition, it allows accurate determination of volumetricproductivity and yield of product formation, two of the most importantparameters in benchmarking strain performance. Fermentations are carriedout in 1-L bioreactors with 600 mL working volume, equipped withtemperature and pH control. The reactor is continuously sparged with N₂at approximately 0.5 L/min to ensure that dissolved oxygen (DO) levelsremain below detection levels. The culture medium is the same asdescribed above, except that the glucose concentration is increased inaccordance with the higher cell density achievable in a fermentationvessel.

Chemostat Testing:

Chemostat experiments are conducted to obtain a direct measure of howthe switch in fermentation mode from batch to continuous affects productyield and volumetric productivity. The bioreactors described above usingbatch mode are operated in chemostat mode through continuous supply ofmedium and removal of spent culture. The inlet flow rate is set tomaintain a constant dilution rate of 80% of the maximum growth rateobserved for each strain in batch, and the outlet flow is controlled tomaintain level. Glucose is the limiting nutrient in the medium, and setto achieve the desired optical density in the vessel.

Adaptive Evolution:

The knockout strains are initially expected to exhibit suboptimal growthrates until their metabolic networks have adjusted to their missingfunctionalities. To allow this adjustment, the strains is adaptivelyevolved. By subjecting the strains to adaptive evolution, cellulargrowth rate becomes the primary selection pressure and the mutant cellsare compelled to reallocate their metabolic fluxes in order to enhancetheir rates of growth. This reprogramming of metabolism has beenrecently demonstrated for several E. coli mutants that had beenadaptively evolved on various substrates to reach the growth ratespredicted a priori by an in silico model (Fong and Palsson, Nat. Genet.36(10):1056-1058 (2004). The OptKnock-generated strains are adaptivelyevolved in triplicate (running in parallel) due to differences in theevolutionary patterns witnessed previously in E. coli (Fong and Palsson,Nat Genet. 36(10):1056-1058 (2004); Fong et al., J. Bacteriol.185(21):6400-6408 (2003); Ibarra et al., Nature 420(6912):186-189(2002)) that could potentially result in one strain having superiorproduction qualities over the others. Evolutions are run for a period of2-6 weeks, depending upon the rate of growth improvement attained. Ingeneral, evolutions are stopped once a stable phenotype is obtained. Thegrowth-coupled biochemical production concept behind the OptKnockapproach results in the generation of genetically stable overproducers.

Although described as deletion sets, it is understood, as disclosedherein, that gene sets can be deleted or disrupted so that encoded geneproduct activities are reduced or eliminated. Thus, the gene deletionsets of Tables 14-16 can be used to delete or disrupt a gene set in ahost organism in which an increased production of 6-ACA, adipate and/orHMDA is desired. It is understood that any of the disclosed genedeletion sets can be used to generate knockout strains with disrupted ordeleted genes that confer increased production of 6-ACA, adipate and/orHMDA.

TABLE 12 A list of all the reaction stoichiometries and the associatedgenes known to be associated with the reactions identified for deletionin the strategies listed in Tables 1 and 2. Reaction AbbreviationReaction Name Reaction Stoichiometry Associated genes ACKr Acetatekinase [c]: ac + atp <==> actp + adp (b3115 or b2296 or b1849) ADHErAlcohol dehydrogenase [c]: accoa + (2) h + (2) nadh (b0356 or b1478 orb1241) <==> coa + etoh + (2) nad AKGD Alpha-ketoglutarate [c]: akg +coa + nad --> co2 + (b0116 and b0726 and b0727) dehydrogenase nadh +succoa ALAR Alanine racemase [c]: ala-L <==> ala-D b4053 ASNS2Asparagine synthetase [c]: asp-L + atp + nh4 --> amp + b3744 asn-L + h +ppi ASPT L-aspartase [c]: asp-L --> fum + nh4 b4139 ATPS4r ATP synthaseadp[c] + (4) h[p] + pi[c] <==> (((b3736 and b3737 and b3738) atp[c] +(3) h[c] + h2o[c] and (b3731 and b3732 and b3733 and b3734 and b3735))or ((b3736 and b3737 and b3738) and (b3731 and b3732 and b3733 and b3734and b3735) and b3739)) CBMK2 Carbamate kinase [c]: atp + co2 + nh4 -->adp + (b0521 or b0323 or b2874) cbp + (2) h DAAD D-amino acid [c]:ala-D + fad + h2o --> fadh2 + b1189 dehydrogenase nh4 + pyr EDA2-dehydro-3-deoxy- [c]: 2ddg6p --> g3p + pyr b1850 phosphogluconatealdolase ENO Enolase [c]: 2pg <==> h2o + pep b2779 FBAFructose-bis-phosphate [c]: fdp <==> dhap + g3p (b2097 or b2925 orb1773) aldolase FRD Fumarate reductase [c]: fum + mql8 --> mqn8 + (b4151and b4152 and b4153 and succ b4154) FUM Fumarase [c]: fum + h2o <==>mal-L (b1612 or b4122 or b1611) G5SD Glutamate-5- [c]: glu5p + h + nadph--> b0243 semialdehyde glu5sa + nadp + pi dehyrogenase G6PDHyGlucose-6-phosphate [c]: g6p + nadp <==> 6pgl + h + b1852 dehydrogenasenadph GLCpts D-glucose transport via glc-D[e] + pep[c] --> g6p[c] +((b2417 and b1101 and b2415 and PTS mechanism pyr[c] b2416) or (b1817and b1818 and b1819 and b2415 and b2416) or (b2417 and b1621 and b2415and b2416)) GLU5K Gluatmate-5-kinase [c]: atp + glu-L --> adp + glu5pb0242 GLUDy Glutamate dehydrogenase [c]: glu-L + h2o + nadp <==> b1761akg + h + nadph + nh4 HEX1 Hexokinase [c]: atp + glc-D --> adp + g6p + hb2388 LDH_D Lactate dehydrogenase [c]: lac-D + nad <==> h + nadh + b1380or b2133 pyr MDH Malate dehydrogenase [c]: mal-L + nad <==> h + nadh +b3236 oaa ME1x Malic enzyme (NAD) [c]: mal-L + nad --> co2 + nadh +b1479 pyr ME2 Malic enzyme (NADP) [c]: mal-L + nadp --> co2 + b2463nadph + pyr NADH12 NADH dehydrogenase [c]: h + nadh + ubq8 --> nad +b1109 (ubiquinone-8) ubq8h2 NADH6 NADH dehydrogenase (4.5) h[c] +nadh[c] + ubq8[c] --> (b2276 and b2277 and b2278 and (ubiquinone-8 and3.5 (3.5) h[e] + nad[c] + b2279 and b2280 and b2281 and protons)ubq8h2[c] b2282 and b2283 and b2284 and b2285 and b2286 and b2287 andb2288) PDH Pyruvate dehydrogenase [c]: coa + nad + pyr --> accoa +((b0114 and b0115 and b0116) or (b0116 co2 + nadh and b0726 and b0727)or (b0116 and b2903 and b2904 and b2905)) PFK Phosphofructokinase [c]:atp + f6p --> adp + fdp + h (b3916 or b1723) PFLi Pyruvate formate lyase[c]: coa + pyr --> accoa + for (((b0902 and b0903) and b2579) or (b0902and b0903) or (b0902 and b3114) or (b3951 and b3952)) PGDHPhosphogluconate [c]: 6pgc + nadp --> co2 + b2029 dehyrogenase nadph +ru5p-D PGDHY Phosphogluconate [c]: 6pgc --> 2ddg6p + h2o b1851dehydratase PGI Glucose-6-phosphate [c]: g6p <==> f6p b4025 isomerasePGL 6- [c]: 6pgl + h2o --> 6pgc + h b0767 Phosphogluconolactonase PGMPhosphoglycerate mutase [c]: 3pg <==> 2pg b3612 PPC Phosphoenolpyruvate[c]: co2 + h2o + pep --> h + oaa + b3956 carboxylase pi PPCKPhosphoenolpyruvate [c]: atp + oaa --> adp + co2 + b3403 carboxykinasepep PRO1z Proline oxidase [c]: fad + pro-L --> 1pyr5c + b1014 fadh2 + hPTAr Phosphotransacetylase [c]: accoa + pi <==> actp + coa b2297 PYKPyruvate kinase [c]: adp + h + pep --> atp + pyr (b1854 or b1676) RPERibulose-5-phosphate-5- [c]: ru5p-D <==> xu5p-D (b4301 or b3386)epimerase SUCD4 Succinate dehydrogenase [c]: fadh2 + ubq8 <==> fad +(b0721 and b0722 and b0723 and ubq8h2 b0724 SUCOAS Succinyl-CoAsynthetase [c]: atp + coa + succ <==> adp + (b0728 and b0729) pi +succoa TAL Transaldoalse [c]: g3p + s7p <==> e4p + f6p (b2464 or b0008)THD2 NADP transhydrogenase (2) h[e] + nadh[c] + nadp[c] --> (b1602 andb1603) (2) h[c] + nad[c] + nadph[c] THD5 NAD transhydrogenase [c]: nad +nadph --> nadh + (b3962 or (b1602 and b1603)) nadp TKT1 Transketolase[c]: r5p + xu5p-D <==> g3p + (b2935 or b2465) s7p TKT2 Transketolase[c]: e4p + xu5p-D <==> f6p + (b2935 or b2465) g3p TPI Triosephosphate[c]: dhap <==> g3p b3919 isomerase VALTA Valine transaminase [c]: akg +val-L <==> 3mob + b3770 glu-L

TABLE 13 List of the metabolite abbreviations, the corresponding namesand locations of all the metabolites that participate in the reactionslisted in Table 12. Metabolite Abbreviation Compartment Metabolite Name13dpg Cytosol 3-Phospho-D-glyceroyl phosphate 1pyr5c Cytosol1-Pyrroline-5-carboxylate 2ddg6p Cytosol 2-Dehydro-3-deoxy-D-gluconate6-phosphate 2pg Cytosol D-Glycerate 2-phosphate 3mob Cytosol3-Methyl-2-oxobutanoate 3pg Cytosol 3-Phospho-D-glycerate 6pgc Cytosol6-Phospho-D-gluconate 6pgl Cytosol 6-phospho-D-glucono-1,5-lactone acCytosol Acetate accoa Cytosol Acetyl-CoA actp Cytosol Acetyl phosphateadp Cytosol Adenosine diphosphate akg Cytosol 2-Oxoglutarate ala-DCytosol D-alanine ala-L Cytosol L-alanine amp Cytosol Adenosinemonophosphate asn-L Cytosol L-asparagine asp-L Cytosol L-aspartate atpCytosol Adenosine triphosphate cbp Cytosol Carbamoyl phosphate co2Cytosol Carbon dioxide coa Cytosol Coenzyme A dha CytosolDihydroxyacetone dhap Cytosol Dihydroxyacetone phosphate e4p CytosolD-Erythrose 4-phosphate etoh Cytosol Ethanol f6p Cytosol D-Fructose6-phosphate fad Cytosol Flavin adenine dinucleotide fadh2 Cytosol Flavinadenine dinucleotide-reduced fdp Cytosol D-Fructose 1,6-bisphosphate forCytosol Formate fum Cytosol Fumarate g3p Cytosol Glyceraldehyde3-phosphate g6p Cytosol D-Glucose 6-phosphate glc-D[e] Extra-organismD-Glucose glu5p Cytosol L-glutamate 5-phosphate glu5sa CytosolL-glutamate 5-semialdehyde glu-L Cytosol L-Glutamate h Cytosol H⁺ h[e]Extra-organism H⁺ h2o Cytosol Water lac-D Cytosol D-Lactate mal-LCytosol L-Malate mql-8 Cytosol Menaquinol-8 mqn-8 Cytosol Menaquinone-8nad Cytosol Nicotinamide adenine dinucleotide nadh Cytosol Nicotinamideadenine dinucleotide - reduced nadp Cytosol Nicotinamide adeninedinucleotide phosphate nadph Cytosol Nicotinamide adenine dinucleotidephosphate - reduced nh4 Cytosol Ammonium o2 Cytosol Oxygen oaa CytosolOxaloacetate pep Cytosol Phosphoenolpyruvate pi Cytosol Phosphate ppiCytosol Diphosphate pyr Cytosol Pyruvate r5p Cytosol alpha-D-Ribose5-phosphate ru5p-D Cytosol D-Ribulose 5-phosphate s7p CytosolSedoheptulose 7-phosphate succ Cytosol Succinate succoa CytosolSuccinyl-CoA ubq8 Cytosol Ubiquinone-8 ubq8h2 Cytosol Ubiquinol-8 val-LCytosol L-valine xu5p-D Cytosol D-Xylulose 5-phosphate

TABLE 14 Knockout strain designs for increased production of 6-ACA,showing yields of 6- ACA and biomass. New Design Num 6ACA. ID KO YieldBiomass Rxnlist 1 1 1.72604 0.38773 ATPS4r 2 1 0.83466 0.26712 PGI 3 25.04234 0.22255 HEX1, PGI 4 2 4.11897 0.24338 EDA and/or PGDHY, PGI 5 23.26272 0.35236 ADHEr, NADH6 6 2 2.8403 0.28275 ADHEr, PPCK 7 2 2.055370.33531 GLCpts, NADH6 8 2 2.0373 0.38161 ATPS4r, RPE 9 2 1.89007 0.3845ATPS4r, TAL 10 2 1.49147 0.26024 PFLi, PGI 11 2 1.11406 0.32382 NADH6,PFLi 12 2 0.95551 0.27773 PFLi, PPCK 13 2 0.89333 0.37885 ADHEr, FUM 142 0.41621 0.38953 ADHEr, HEX1 15 2 0.31773 0.36065 ASPT, FUM 16 36.93034 0.18126 EDA and/or PGDHY, NADH6, PGI 17 3 6.91845 0.19552 HEX1,PFLi, PGI 18 3 6.14899 0.1758 EDA and/or PGDHY, PFLi, PGI 19 3 5.814220.16481 ATPS4r, EDA and/or PGDHY, PGI 20 3 5.71646 0.21908 ADHEr, HEX1,PGI 21 3 4.83364 0.29669 ADHEr, NADH6, PFLi 22 3 4.23803 0.24209 ACKrand/or PTAr, EDA and/or PGDHY, PGI 23 3 4.02855 0.29483 ADHEr, MDH, THD2and/or GLUDy 24 3 3.77533 0.25553 ADHEr, ASPT, MDH 25 3 3.65248 0.19372ADHEr, NADH6, PGI 26 3 3.47283 0.25194 ADHEr, NADH6, PPCK 27 3 3.393190.29001 ADHEr, MDH, NADH6 28 3 3.35305 0.34906 ADHEr, NADH6, RPE 29 33.23462 0.28717 ACKr and/or PTAr, ATPS4r, SUCOAS 30 3 3.0877 0.16431ADHEr, PGI, PPCK 31 3 2.90019 0.24841 ADHEr, GLCpts, PPCK 32 3 2.898550.2815 ADHEr, PPCK, RPE 33 3 2.88617 0.25645 ADHEr, FUM, THD2 and/orGLUDy 34 3 2.72186 0.35068 ADHEr, FUM, HEX1 35 3 2.60615 0.3202 ATPS4r,HEX1, PFLi 36 3 2.54001 0.22798 PFLi, PGDH, PGI 37 3 2.5259 0.22921PFLi, PGI, TAL 38 3 2.5129 0.23034 PFLi, PGI, RPE 39 3 2.50442 0.16853ATPS4r, PFLi, PGI 40 3 2.38919 0.18418 GLCpts, NADH6, PGI 41 3 2.307410.33343 ATPS4r, GLCpts, NADH6 42 3 2.16995 0.33092 GLCpts, NADH6, RPE 433 2.11568 0.333 GLCpts, NADH6, TAL 44 3 2.10576 0.25488 ATPS4r, PPCK,PYK 45 3 1.5933 0.25891 FUM, PFLi, PGI 46 3 1.25154 0.3194 NADH6, PFLi,RPE 47 3 1.23093 0.32359 ACKr and/or PTAr, NADH6, PFLi 48 3 1.186430.32149 NADH6, PFLi, TAL 49 3 1.08143 0.27599 PFLi, PPCK, RPE 50 31.0527 0.27673 ACKr and/or PTAr, PFLi, PPCK 51 3 1.03356 0.34314 FUM,HEX1, PFLi 52 3 1.02156 0.27682 PFLi, PPCK, TAL 53 3 0.8538 0.24817 MDH,PFLi, THD2 and/or GLUDy 54 3 0.5753 0.38695 ADHEr, HEX1, RPE 55 30.49968 0.38818 ADHEr, HEX1, TAL 56 3 0.31402 0.35643 FDH2, FUM, NADH657 3 0.23944 0.28074 FUM, PFLi, THD2 and/or GLUDy 58 3 0.23418 0.26581ASPT, MDH, PFLi 59 3 0.20901 0.23724 ASPT, MDH, PYK 60 4 7.08404 0.12641EDA and/or PGDHY, NADH6, PFLi, PGI 61 4 7.0245 0.10838 EDA and/or PGDHY,PFLi, PGI, PPCK 62 4 6.92332 0.16482 ADHEr, HEX1, NADH6, PGI 63 46.89839 0.18171 ACKr and/or PTAr, ADHEr, NADH6, PGI 64 4 6.58534 0.16513EDA and/or PGDHY, GLCpts, PFLi, PGI 65 4 6.36357 0.11937 EDA and/orPGDHY, PFLi, PGI, THD2 and/or GLUDy 66 4 6.22082 0.11375 ATPS4r, EDAand/or PGDHY, PFLi, PGI 67 4 5.8701 0.13379 ADHEr, EDA and/or PGDHY,PGI, PPCK 68 4 5.85757 0.23874 ADHEr, MDH, NADH6, THD2 and/or GLUDy 69 45.85411 0.19685 ADHEr, HEX1, PGI, PPS 70 4 5.67774 0.13549 EDA and/orPGDHY, PGI, PPCK, PYK 71 4 5.46506 0.26673 EDA and/or PGDHY, MDH, PFLi,THD2 and/or GLUDy 72 4 5.40131 0.26362 ADHEr, MDH, PFLi, THD2 and/orGLUDy 73 4 5.08219 0.13778 ATPS4r, NADH6, PFLi, PGI 74 4 4.88764 0.27849ADHEr, NADH12, NADH6, PFLi 75 4 4.88489 0.27942 ADHEr, FUM, NADH6, PFLi76 4 4.81778 0.26037 ADHEr, ATPS4r, MDH, NADH6 77 4 4.73419 0.21859ADHEr, NADH6, PPCK, THD2 and/or GLUDy 78 4 4.63783 0.29595 ADHEr,ATPS4r, FDH2, NADH6 79 4 4.51525 0.21818 ADHEr, FUM, PPCK, THD2 and/orGLUDy 80 4 4.51525 0.21818 ADHEr, MDH, PPCK, THD2 and/or GLUDy 81 44.30547 0.19131 ADHEr, ATPS4r, NADH6, PGI 82 4 4.2733 0.23078 ADHEr,ATPS4r, NADH6, PPCK 83 4 4.16417 0.18771 ATPS4r, NADH6, PGI, RPE 84 44.16053 0.18697 ATPS4r, NADH6, PGI, TAL 85 4 4.15658 0.18617 ATPS4r,NADH6, PGDH, PGI 86 4 4.12032 0.32701 ADHEr, ATPS4r, FUM, NADH6 87 43.9718 0.23354 ADHEr, ATPS4r, MDH, PPCK 88 4 3.9718 0.23354 ADHEr,ATPS4r, FUM, PPCK 89 4 3.8747 0.21758 ADHEr, ASPT, GLCpts, MDH 90 43.84814 0.25342 ADHEr, ASPT, MDH, RPE 91 4 3.83986 0.2047 ADHEr, ASPT,MDH, PYK 92 4 3.75472 0.32987 ADHEr, ATPS4r, HEX1, NADH6 93 4 3.549650.29114 ADHEr, ATPS4r, MDH, PGDH 94 4 3.54605 0.21695 ADHEr, GLCpts,NADH6, PPCK 95 4 3.54385 0.218 ADHEr, NADH6, PPCK, PYK 96 4 3.536150.25027 ADHEr, NADH6, PPCK, RPE 97 4 3.5018 0.32809 ADHEr, ATPS4r, FUM,HEX1 98 4 3.46904 0.25375 ADHEr, GLCpts, MDH, NADH6 99 4 3.46528 0.28851ADHEr, MDH, NADH6, RPE 100 4 3.44916 0.13425 ADHEr, PFLi, PGI, PPCK 1014 3.44555 0.26498 ADHEr, HEX1, NADH6, THD2 and/or GLUDy 102 4 3.437760.29402 ADHEr, ATPS4r, MDH, TAL 103 4 3.36596 0.19156 FUM, NADH6, PGI,THD5 104 4 3.36596 0.19156 MDH, NADH6, PGI, THD5 105 4 3.33377 0.2967ADHEr, ATPS4r, MDH, RPE 106 4 3.33152 0.33978 ADHEr, FUM, NADH6, TAL 1074 3.32935 0.34088 ADHEr, HEX1, NADH6, TAL 108 4 3.32788 0.32122 ADHEr,FUM, HEX1, NADH6 109 4 3.31278 0.3493 ADHEr, GLU5K, NADH6, TAL 110 43.31278 0.3493 ADHEr, G5SD, NADH6, TAL 111 4 3.17484 0.10602 ADHEr, PGI,PPCK, THD2 and/or GLUDy 112 4 3.16614 0.11184 ADHEr, MDH, PGI, THD2and/or GLUDy 113 4 3.16614 0.11184 ADHEr, FUM, PGI, THD2 and/or GLUDy114 4 3.11125 0.24826 ADHEr, ATPS4r, PPCK, THD2 and/or GLUDy 115 42.96628 0.21051 ADHEr, LDH_D, MDH, THD2 and/or GLUDy 116 4 2.955290.24477 ADHEr, PPCK, RPE, THD2 and/or GLUDy 117 4 2.95136 0.24731 ADHEr,GLCpts, PPCK, RPE 118 4 2.94249 0.25305 ADHEr, FUM, RPE, THD2 and/orGLUDy 119 4 2.93765 0.22693 ADHEr, MDH, PPCK, PYK 120 4 2.93765 0.22693ADHEr, FUM, PPCK, PYK 121 4 2.9332 0.24406 ADHEr, PPCK, TAL, THD2 and/orGLUDy 122 4 2.90913 0.24328 ADHEr, PGDH, PPCK, THD2 and/or GLUDy 123 42.90913 0.24328 ADHEr, PGL and/or G6PDHy, PPCK, THD2 and/or GLUDy 124 42.90081 0.26381 ADHEr, FUM, PPCK, TAL 125 4 2.90081 0.26381 ADHEr, MDH,PPCK, TAL 126 4 2.86334 0.28161 ATPS4r, GLCpts, NADH6, PFLi 127 42.79498 0.34856 ADHEr, FUM, HEX1, RPE 128 4 2.7362 0.31504 ATPS4r, HEX1,PFLi, RPE 129 4 2.70922 0.18179 ATPS4r, GLCpts, NADH6, PGI 130 4 2.674690.31748 ATPS4r, HEX1, PFLi, TAL 131 4 2.61943 0.36027 ADHEr, HEX1, PFLi,PPS 132 4 2.60629 0.2178 PFLi, PGDH, PGI, TAL 133 4 2.60314 0.31113ADHEr, HEX1, MDH, PFLi 134 4 2.56456 0.18551 ASPT, NADH6, PGI, THD5 1354 2.55949 0.17944 ATPS4r, PFLi, PPCK, PYK 136 4 2.45433 0.3546 ADHEr,ATPS4r, HEX1, THD2 and/or GLUDy 137 4 2.45166 0.03625 GLCpts, NADH6,PPCK, PYK 138 4 2.44258 0.32891 ATPS4r, GLCpts, NADH6, RPE 139 4 2.439390.25153 ATPS4r, PPCK, PYK, RPE 140 4 2.37857 0.33105 ATPS4r, GLCpts,NADH6, TAL 141 4 2.30961 0.22691 ATPS4r, NADH6, PPCK, PYK 142 4 2.281350.25311 ATPS4r, PPCK, PYK, TAL 143 4 2.18633 0.3609 ADHEr, HEX1, PPS,THD2 and/or GLUDy 144 4 2.11347 0.31979 ADHEr, ATPS4r, HEX1, MDH 145 42.05497 0.19746 ASPT, ATPS4r, PGI, THD5 146 4 1.83108 0.25754 MDH, PFLi,PGI, THD2 and/or GLUDy 147 4 1.65594 0.25767 ACKr and/or PTAr, FUM,PFLi, PGI 148 4 1.36673 0.31918 ACKr and/or PTAr, NADH6, PFLi, RPE 149 41.30242 0.32127 ACKr and/or PTAr, NADH6, PFLi, TAL 150 4 1.28243 0.32216ACKr and/or PTAr, GLU5K, NADH6, PFLi 151 4 1.28243 0.32216 ACKr and/orPTAr, G5SD, NADH6, PFLi 152 4 1.26654 0.34191 ACKr and/or PTAr, FUM,HEX1, PFLi 153 4 1.18926 0.34046 FUM, HEX1, PFLi, RPE 154 4 1.177560.275 ACKr and/or PTAr, PFLi, PPCK, RPE 155 4 1.1182 0.27583 ACKr and/orPTAr, PFLi, PPCK, TAL 156 4 1.1153 0.34173 FUM, HEX1, PFLi, TAL 157 40.95889 0.24471 MDH, PFLi, RPE, THD2 and/or GLUDy 158 4 0.9475 0.3633ADHEr, FUM, PGL and/or G6PDHy, TAL 159 4 0.9475 0.3633 ADHEr, FUM, PGDH,TAL 160 4 0.90913 0.24635 MDH, PFLi, TAL, THD2 and/or GLUDy 161 40.36773 0.36076 HEX1, PFLi, PPS, THD2 and/or GLUDy 162 4 0.3476 0.2132ADHEr, MDH, RPE, THD2 and/or GLUDy 163 4 0.32915 0.27571 FUM, PFLi, RPE,THD2 and/or GLUDy 164 5 6.98742 0.06748 ADHEr, EDA and/or PGDHY, NADH6,PGI, THD2 and/or GLUDy 165 5 6.97749 0.0848 ACKr and/or PTAr, ATPS4r,HEX1, PFLi, PGI 166 5 6.70882 0.01277 EDA and/or PGDHY, FUM, PFLi, PGI,THD2 and/or GLUDy 167 5 6.70882 0.01277 EDA and/or PGDHY, MDH, PFLi,PGI, THD2 and/or GLUDy 168 5 6.69134 0.13239 ADHEr, ASPT, ATPS4r,GLCpts, MDH 169 5 6.55123 0.09841 ADHEr, ASPT, MDH, PGL and/or G6PDHy,PYK 170 5 6.55123 0.09841 ADHEr, ASPT, EDA and/or PGDHY, MDH, PYK 171 56.3052 0.17034 ADHEr, ASPT, ATPS4r, MDH, PGL and/or G6PDHy 172 5 6.208710.17434 EDA and/or PGDHY, MDH, PFLi, PGI, THD5 173 5 6.12639 0.08557ADHEr, FRD and/or SUCD4, LDH_D, MDH, THD2 and/or GLUDy 174 5 6.033860.19348 ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH 175 5 5.98628 0.0693ATPS4r, EDA and/or PGDHY, PGI, PPCK, PYK 176 5 5.98165 0.07186 ADHEr,ATPS4r, EDA and/or PGDHY, PGI, PPCK 177 5 5.93527 0.09761 ADHEr, EDAand/or PGDHY, GLCpts, PGI, PPCK 178 5 5.87444 0.0434 ADHEr, EDA and/orPGDHY, MDH, PGI, THD2 and/or GLUDy 179 5 5.87444 0.0434 ADHEr, EDAand/or PGDHY, FUM, PGI, THD2 and/or GLUDy 180 5 5.82336 0.16439 ACKrand/or PTAr, ADHEr, ASPT, MDH, PYK 181 5 5.75418 0.21654 ACKr and/orPTAr, ADHEr, LDH_D, MDH, NADH6 182 5 5.65488 0.26204 ACKr and/or PTAr,ADHEr, MDH, PFLi, THD2 and/or GLUDy 183 5 5.59555 0.20952 ADHEr, ATPS4r,GLCpts, MDH, NADH6 184 5 5.30614 0.21123 ADHEr, ASPT, LDH_D, MDH, THD2and/or GLUDy 185 5 5.21058 0.214 ADHEr, ASPT, ATPS4r, LDH_D, MDH 186 55.20451 0.17174 ADHEr, LDH_D, NADH6, PFLi, PPCK 187 5 5.1776 0.13724ADHEr, NADH12, NADH6, PFLi, PGI 188 5 5.11744 0.13758 ADHEr, MDH, NADH6,PFLi, PGI 189 5 5.11744 0.13758 ADHEr, FUM, NADH6, PFLi, PGI 190 55.11714 0.20117 ADHEr, LDH_D, MDH, PFLi, PPCK 191 5 5.11714 0.20117ADHEr, FUM, LDH_D, PFLi, PPCK 192 5 5.10437 0.12227 ADHEr, FUM, PFLi,PGI, PPCK 193 5 5.10437 0.12227 ADHEr, MDH, PFLi, PGI, PPCK 194 55.09877 0.17589 ADHEr, ATPS4r, GLCpts, NADH6, PPCK 195 5 5.0606 0.22022ADHEr, LDH_D, MDH, NADH6, PFLi 196 5 5.02693 0.21085 ADHEr, ATPS4r,NADH6, PGDH, PPCK 197 5 5.02693 0.21085 ADHEr, ATPS4r, NADH6, PGL and/orG6PDHy, PPCK 198 5 5.00057 0.21154 ADHEr, ATPS4r, NADH6, PPCK, TAL 199 54.97638 0.21218 ADHEr, ATPS4r, NADH6, PPCK, RPE 200 5 4.88781 0.27844ADHEr, HEX1, LDH_D, NADH6, PFLi 201 5 4.88512 0.29281 ADHEr, GLU5K,NADH6, PFLi, RPE 202 5 4.88512 0.29281 ADHEr, G5SD, NADH6, PFLi, RPE 2035 4.885 0.29286 ADHEr, ASNS2, NADH6, PFLi, RPE 204 5 4.85776 0.29446ADHEr, ATPS4r, FDH2, NADH6, RPE 205 5 4.83644 0.29517 ADHEr, ATPS4r,FDH2, NADH6, TAL 206 5 4.7175 0.34071 ADHEr, FUM, LDH_D, PFLi, THD2and/or GLUDy 207 5 4.66386 0.18074 ADHEr, ATPS4r, GLCpts, MDH, PPCK 2085 4.66386 0.18074 ADHEr, ATPS4r, FUM, GLCpts, PPCK 209 5 4.63095 0.20189ADHEr, ATPS4r, GLCpts, PFLi, PPCK 210 5 4.49707 0.25782 ADHEr, ATPS4r,EDA and/or PGDHY, MDH, THD2 and/or GLUDy 211 5 4.4958 0.1778 ADHEr,ASPT, MDH, NADH6, PYK 212 5 4.41977 0.25646 ADHEr, ATPS4r, GLCpts, MDH,THD2 and/or GLUDy 213 5 4.29965 0.18828 ADHEr, ASPT, ATPS4r, MDH, PYK214 5 4.29026 0.24034 ACKr and/or PTAr, EDA and/or PGDHY, GLU5K, GLYCL,PGI 215 5 4.29026 0.24034 ACKr and/or PTAr, EDA and/or PGDHY, G5SD,GLYCL, PGI 216 5 4.28287 0.11665 ACKr and/or PTAr, ATPS4r, PPCK, PYK,SUCOAS 217 5 4.24299 0.26303 ACKr and/or PTAr, ADHEr, HEX1, LDH_D, NADH6218 5 4.18509 0.32432 ADHEr, ATPS4r, HEX1, NADH6, PGL and/or G6PDHy 2195 4.18509 0.32432 ADHEr, ATPS4r, HEX1, NADH6, PGDH 220 5 4.15013 0.1124ADHEr, ASPT, MDH, PYK, THD2 and/or GLUDy 221 5 4.14582 0.32483 ADHEr,ATPS4r, HEX1, NADH6, TAL 222 5 4.1099 0.32529 ADHEr, ATPS4r, HEX1,NADH6, RPE 223 5 4.04473 0.12883 ADHEr, PFLi, PGDH, PGI, PPCK 224 54.03844 0.12934 ADHEr, PFLi, PGI, PPCK, TAL 225 5 4.03266 0.12981 ADHEr,PFLi, PGI, PPCK, RPE 226 5 4.02531 0.1111 ADHEr, GLCpts, PFLi, PGI, PPCK227 5 3.95205 0.10694 ACKr and/or PTAr, ATPS4r, PGDH, PGI, SUCOAS 228 53.94203 0.1079 ACKr and/or PTAr, ATPS4r, PGI, SUCOAS, TAL 229 5 3.93670.21579 ADHEr, ASPT, GLCpts, MDH, RPE 230 5 3.93273 0.1088 ACKr and/orPTAr, ATPS4r, PGI, RPE, SUCOAS 231 5 3.88257 0.21457 ADHEr, ASPT, LDH_D,MDH, PPCK 232 5 3.84571 0.22865 ADHEr, ASPT, LDH_D, MDH, NADH6 233 53.76049 0.30843 ADHEr, ATPS4r, FUM, HEX1, PFLi 234 5 3.74674 0.21738ADHEr, ATPS4r, LDH_D, PFLi, PPCK 235 5 3.71345 0.27754 ADHEr, ATPS4r,MDH, PGDH, TAL 236 5 3.69299 0.14673 ADHEr, NADH6, PPCK, PYK, THD2and/or GLUDy 237 5 3.64625 0.1409 ACKr and/or PTAr, ADHEr, FUM, PGI,PPCK 238 5 3.64625 0.1409 ACKr and/or PTAr, ADHEr, MDH, PGI, PPCK 239 53.60057 0.21551 ADHEr, GLCpts, NADH6, PPCK, RPE 240 5 3.59735 0.21725ADHEr, NADH6, PPCK, PYK, RPE 241 5 3.59622 0.19297 ADHEr, GLCpts, MDH,NADH6, PPCK 242 5 3.59622 0.19297 ADHEr, FUM, GLCpts, NADH6, PPCK 243 53.59594 0.19311 ADHEr, MDH, NADH6, PPCK, PYK 244 5 3.59594 0.19311ADHEr, FUM, NADH6, PPCK, PYK 245 5 3.59157 0.1952 ADHEr, FUM, GLCpts,NADH6, THD2 and/or GLUDy 246 5 3.57086 0.18962 ATPS4r, FUM, NADH6, PGI,THD5 247 5 3.57086 0.18962 ATPS4r, MDH, NADH6, PGI, THD5 248 5 3.552040.22769 ADHEr, FUM, NADH6, PPCK, TAL 249 5 3.55204 0.22769 ADHEr, MDH,NADH6, PPCK, TAL 250 5 3.53212 0.25244 ADHEr, GLCpts, MDH, NADH6, RPE251 5 3.52326 0.22785 ADHEr, FUM, GLU5K, NADH6, PPCK 252 5 3.523260.22785 ADHEr, G5SD, MDH, NADH6, PPCK 253 5 3.52326 0.22785 ADHEr,GLU5K, MDH, NADH6, PPCK 254 5 3.52326 0.22785 ADHEr, FUM, G5SD, NADH6,PPCK 255 5 3.52319 0.22788 ADHEr, ASNS2, FUM, NADH6, PPCK 256 5 3.523190.22788 ADHEr, ASNS2, MDH, NADH6, PPCK 257 5 3.51328 0.26261 ADHEr,HEX1, NADH6, RPE, THD2 and/or GLUDy 258 5 3.40958 0.31856 ADHEr, FUM,HEX1, NADH6, RPE 259 5 3.19551 0.34972 ADHEr, ATPS4r, HEX1, PPS, THD2and/or GLUDy 260 5 3.04993 0.16253 ADHEr, FUM, LDH_D, PPCK, THD2 and/orGLUDy 261 5 3.04993 0.16253 ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy262 5 3.02516 0.17674 ADHEr, GLCpts, LDH_D, MDH, THD2 and/or GLUDy 263 52.95797 0.22895 ADHEr, GLCpts, MDH, PPCK, TAL 264 5 2.95797 0.22895ADHEr, FUM, GLCpts, PPCK, TAL 265 5 2.94768 0.30664 ADHEr, ATPS4r, HEX1,MDH, PFLi 266 5 2.93267 0.03242 ATPS4r, GLCpts, NADH6, PPCK, PYK 267 52.93156 0.27787 ATPS4r, GLCpts, NADH6, PFLi, RPE 268 5 2.92149 0.26881ADHEr, ATPS4r, MDH, PGL and/or G6PDHy, PPS 269 5 2.76831 0.35787 ADHEr,HEX1, PFLi, PPS, RPE 270 5 2.73201 0.30945 ADHEr, HEX1, MDH, PFLi, RPE271 5 2.69754 0.35901 ADHEr, HEX1, PFLi, PPS, TAL 272 5 2.69256 0.22448ATPS4r, NADH6, PPCK, PYK, RPE 273 5 2.67071 0.31025 ADHEr, HEX1, MDH,PFLi, TAL 274 5 2.63273 0.17703 ATPS4r, PFLi, PPCK, PYK, RPE 275 52.61965 0.35455 ADHEr, ATPS4r, HEX1, RPE, THD2 and/or GLUDy 276 5 2.54790.2938 ADHEr, ATPS4r, GLUDy, HEX1, THD2 and/or GLUDy 277 5 2.54080.35457 ADHEr, ATPS4r, HEX1, TAL, THD2 and/or GLUDy 278 5 2.510930.22563 ATPS4r, NADH6, PPCK, PYK, TAL 279 5 2.50239 0.09117 GLCpts,PFLi, PGI, PPCK, PYK 280 5 1.97671 0.25583 ACKr and/or PTAr, MDH, PFLi,PGI, THD2 and/or GLUDy 281 5 1.41987 0.33925 ACKr and/or PTAr, FUM,HEX1, PFLi, RPE 282 5 1.41694 0.31779 ACKr and/or PTAr, GLU5K, NADH6,PFLi, RPE 283 5 1.41694 0.31779 ACKr and/or PTAr, G5SD, NADH6, PFLi, RPE284 5 1.35397 0.34014 ACKr and/or PTAr, FUM, GLU5K, HEX1, PFLi 285 51.35397 0.34014 ACKr and/or PTAr, FUM, G5SD, HEX1, PFLi 286 5 1.353240.31986 ACKr and/or PTAr, GLU5K, NADH6, PFLi, TAL 287 5 1.35324 0.31986ACKr and/or PTAr, G5SD, NADH6, PFLi, TAL 288 5 1.34703 0.34051 ACKrand/or PTAr, FUM, HEX1, PFLi, TAL 289 5 1.16644 0.24173 ADHEr, FRDand/or SUCD4, LDH_D, PPS, THD2 and/or GLUDy 290 5 0.935 0.24579 ACKrand/or PTAr, ACS, MDH, PFLi, THD2 and/or GLUDy 291 5 0.5376 0.11141ADHEr, FRD and/or SUCD4, GLUDy, LDH_D, THD2 and/or GLUDy 292 5 0.441450.36033 HEX1, PFLi, PPS, RPE, THD2 and/or GLUDy 293 5 0.41407 0.27855ACKr and/or PTAr, ACS, FUM, PFLi, THD2 and/or GLUDy 294 5 0.341530.21403 ACKr and/or PTAr, ADHEr, MDH, TAL, THD2 and/or GLUDy 295 50.29506 0.27092 FUM, PFLi, PGDH, TAL, THD2 and/or GLUDy 296 5 0.295060.27092 FUM, PFLi, PGL and/or G6PDHy, TAL, THD2 and/or GLUDy 297 50.29233 0.27766 FUM, GLYCL, PFLi, TAL, THD2 and/or GLUDy 298 2 1.726040.38773 ADHEr, ATPS4r 299 2 0.83466 0.26712 ADHEr, PGI 300 3 4.118970.24338 ADHEr, EDA and/or PGDHY, PGI 301 3 2.0373 0.38161 ADHEr, ATPS4r,RPE 302 3 1.89007 0.3845 ADHEr, ATPS4r, TAL 303 3 1.49147 0.26024 ADHEr,PFLi, PGI 304 4 6.93034 0.18126 ADHEr, EDA and/or PGDHY, NADH6, PGI 3054 6.91845 0.19552 ADHEr, HEX1, PFLi, PGI 306 4 6.14899 0.1758 ADHEr, EDAand/or PGDHY, PFLi, PGI 307 4 5.81422 0.16481 ADHEr, ATPS4r, EDA and/orPGDHY, PGI 308 4 4.23803 0.24209 ACKr and/or PTAr, ADHEr, EDA and/orPGDHY, PGI 309 4 3.23462 0.28717 ACKr and/or PTAr, ADHEr, ATPS4r, SUCOAS310 4 2.60615 0.3202 ADHEr, ATPS4r, HEX1, PFLi 311 4 2.54001 0.22798ADHEr, PFLi, PGDH, PGI 312 4 2.5259 0.22921 ADHEr, PFLi, PGI, TAL 313 42.5129 0.23034 ADHEr, PFLi, PGI, RPE 314 4 2.50442 0.16853 ADHEr,ATPS4r, PFLi, PGI 315 4 1.5933 0.25891 ADHEr, FUM, PFLi, PGI 316 57.08404 0.12641 ADHEr, EDA and/or PGDHY, NADH6, PFLi, PGI 317 5 7.02450.10838 ADHEr, EDA and/or PGDHY, PFLi, PGI, PPCK 318 5 6.58534 0.16513ADHEr, EDA and/or PGDHY, GLCpts, PFLi, PGI 319 5 6.36357 0.11937 ADHEr,EDA and/or PGDHY, PFLi, PGI, THD2 and/or GLUDy 320 5 6.22082 0.11375ADHEr, ATPS4r, EDA and/or PGDHY, PFLi, PGI 321 5 5.08219 0.13778 ADHEr,ATPS4r, NADH6, PFLi, PGI 322 5 2.7362 0.31504 ADHEr, ATPS4r, HEX1, PFLi,RPE 323 5 2.67469 0.31748 ADHEr, ATPS4r, HEX1, PFLi, TAL 324 5 2.606290.2178 ADHEr, PFLi, PGDH, PGI, TAL 325 5 2.05497 0.19746 ADHEr, ASPT,ATPS4r, PGI, THD5 326 5 1.65594 0.25767 ACKr and/or PTAr, ADHEr, FUM,PFLi, PGI 327 6 7.82999 0.02101 ADHEr, ASPT, LDH_D, MDH, PFLi, THD2and/or GLUDy 328 6 7.21161 0.05631 ADHEr, EDA and/or PGDHY, HEX1, NADH6,PGI, THD2 and/or GLUDy 329 6 7.16447 0.10326 ADHEr, ASPT, LDH_D, MDH,PFLi, PYK 330 6 6.97749 0.0848 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1,PFLi, PGI 331 6 6.89088 0.16538 ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGLand/or G6PDHy 332 6 6.86543 0.14022 ADHEr, ASPT, ATPS4r, LDH_D, MDH,PFLi 333 6 6.72704 0.08076 ADHEr, ASPT, EDA and/or PGDHY, MDH, NADH6,PYK 334 6 6.72704 0.08076 ADHEr, ASPT, MDH, NADH6, PGL and/or G6PDHy,PYK 335 6 6.67303 0.15616 ADHEr, LDH_D, MDH, PFLi, PPCK, THD2 and/orGLUDy 336 6 6.67303 0.15616 ADHEr, FUM, LDH_D, PFLi, PPCK, THD2 and/orGLUDy 337 6 6.62564 0.17535 ADHEr, LDH_D, MDH, NADH6, PFLi, THD2 and/orGLUDy 338 6 6.44535 0.24832 ADHEr, FUM, HEX1, LDH_D, PFLi, THD2 and/orGLUDy 339 6 6.44535 0.24832 ADHEr, HEX1, LDH_D, MDH, PFLi, THD2 and/orGLUDy 340 6 6.3515 0.04992 ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH,THD2 and/or GLUDy 341 6 6.3052 0.17034 ADHEr, ASPT, ATPS4r, EDA and/orPGDHY, MDH, PGDH 342 6 6.25221 0.15227 ACKr and/or PTAr, ADHEr, ASPT,GLCpts, LDH_D, MDH 343 6 6.22508 0.18792 ADHEr, ATPS4r, LDH_D, MDH,NADH6, PFLi 344 6 6.20871 0.17434 ADHEr, EDA and/or PGDHY, MDH, PFLi,PGI, THD5 345 6 6.19852 0.09326 ADHEr, ASPT, FUM, LDH_D, MDH, THD2and/or GLUDy 346 6 6.19408 0.16324 ACKr and/or PTAr, ADHEr, ASPT, LDH_D,MDH, PYK 347 6 6.19255 0.17194 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY,MDH, TAL 348 6 6.18674 0.18098 ACKr and/or PTAr, ADHEr, ASPT, LDH_D,MDH, NADH6 349 6 6.18358 0.11862 ADHEr, ATPS4r, GLCpts, MDH, NADH6, PFLi350 6 6.12313 0.19075 ACKr and/or PTAr, ADHEr, FDH2, LDH_D, MDH, NADH6351 6 6.09846 0.03275 ADHEr, ASPT, LDH_D, MALS, MDH, THD2 and/or GLUDy352 6 6.09846 0.03275 ADHEr, ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy353 6 6.08794 0.17343 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, MDH, RPE354 6 6.01307 0.17456 ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6 355 66.00837 0.14517 ADHEr, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy 356 65.99004 0.18339 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, NADH6 357 65.9797 0.19373 ACKr and/or PTAr, ACS, ADHEr, HEX1, PGI, PPS 358 65.97334 0.11266 ADHEr, GLCpts, MDH, NADH6, PFLi, PGI 359 6 5.966430.16387 ACKr and/or PTAr, ADHEr, ASPT, MDH, PYK, RPE 360 6 5.89830.16412 ACKr and/or PTAr, ADHEr, ASPT, MDH, PYK, TAL 361 6 5.897770.06803 ADHEr, ATPS4r, EDA and/or PGDHY, HEX1, PGI, THD2 and/or GLUDy362 6 5.81808 0.13048 ADHEr, ATPS4r, MDH, NADH6, PFLi, PGI 363 6 5.773130.13132 ADHEr, ATPS4r, NADH12, NADH6, PFLi, PGI 364 6 5.75832 0.26044ACKr and/or PTAr, ADHEr, GLU5K, MDH, PFLi, THD2 and/or GLUDy 365 65.75832 0.26044 ACKr and/or PTAr, ADHEr, G5SD, MDH, PFLi, THD2 and/orGLUDy 366 6 5.75556 0.13165 ADHEr, ATPS4r, FUM, NADH6, PFLi, PGI 367 65.68405 0.15589 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPCK 368 6 5.645180.25081 ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy 369 6 5.614740.1274 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PPS, THD2 and/or GLUDy 3706 5.57706 0.11542 ADHEr, ATPS4r, GLCpts, NADH6, PFLi, THD2 and/or GLUDy371 6 5.55724 0.20645 ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6 372 65.39028 0.01098 ADHEr, ASPT, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 373 65.36336 0.12039 ADHEr, ASPT, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy 374 65.35114 0.12824 ADHEr, NADH12, NADH6, PFLi, PGI, RPE 375 6 5.349920.12867 ADHEr, FUM, NADH6, PFLi, PGI, RPE 376 6 5.34421 0.12775 ADHEr,NADH12, NADH6, PFLi, PGI, TAL 377 6 5.34298 0.12817 ADHEr, FUM, NADH6,PFLi, PGI, TAL 378 6 5.33668 0.12721 ADHEr, NADH12, NADH6, PFLi, PGDH,PGI 379 6 5.33544 0.12763 ADHEr, FUM, NADH6, PFLi, PGDH, PGI 380 65.3133 0.13509 ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK 381 6 5.301210.14588 ADHEr, ATPS4r, GLCpts, NADH6, PPCK, RPE 382 6 5.29298 0.14544ADHEr, ATPS4r, GLCpts, NADH6, PPCK, TAL 383 6 5.28823 0.16642 ADHEr,ASPT, ATPS4r, LDH_D, MDH, PPCK 384 6 5.28401 0.14496 ADHEr, ATPS4r,GLCpts, NADH6, PGL and/or G6PDHy, PPCK 385 6 5.28401 0.14496 ADHEr,ATPS4r, GLCpts, NADH6, PGDH, PPCK 386 6 5.28388 0.145 ADHEr, LDH_D,NADH6, PFLi, PPCK, PYK 387 6 5.23479 0.26578 ADHEr, ATPS4r, HEX1, LDH_D,NADH6, PFLi 388 6 5.22487 0.16488 ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK389 6 5.22487 0.16488 ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK 390 65.18966 0.19912 ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPCK 391 6 5.189660.19912 ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK 392 6 5.17671 0.18111ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi 393 6 5.17275 0.18244 ADHEr,GLCpts, LDH_D, MDH, NADH6, PFLi 394 6 5.16547 0.14992 ADHEr, ASPT,ATPS4r, EDA and/or PGDHY, MDH, PFLi 395 6 5.14231 0.02949 ADHEr, FRDand/or SUCD4, GLUDy, HEX1, LDH_D, THD2 and/or GLUDy 396 6 5.1335 0.31969ADHEr, HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy 397 6 5.12241 0.20902ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE 398 6 5.11384 0.20727 ADHEr,ATPS4r, LDH_D, MDH, NADH6, TAL 399 6 5.1046 0.2054 ADHEr, ATPS4r, LDH_D,MDH, NADH6, PGDH 400 6 5.00987 0.10652 ADHEr, ATPS4r, LDH_D, MDH, NADH6,THD2 and/or GLUDy 401 6 4.99324 0.24292 ADHEr, HEX1, LDH_D, NADH12,NADH6, PFLi 402 6 4.98859 0.24448 ADHEr, FUM, HEX1, LDH_D, NADH6, PFLi403 6 4.91516 0.18237 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK 404 64.91516 0.18237 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK 405 6 4.909970.20313 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, THD2 and/or GLUDy 406 64.86469 0.20078 ADHEr, ATPS4r, GLCpts, PFLi, PPCK, RPE 407 6 4.799880.22937 ACKr and/or PTAr, ADHEr, FUM, MDH, NADH6, PYK 408 6 4.759980.22327 ADHEr, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy 409 6 4.759980.22327 ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/or GLUDy410 6 4.75352 0.20131 ADHEr, ATPS4r, GLCpts, PFLi, PPCK, TAL 411 64.72575 0.27672 ADHEr, ATPS4r, FDH2, HEX1, LDH_D, NADH6 412 6 4.684680.22968 ADHEr, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy 413 6 4.672030.24577 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or GLUDy414 6 4.6646 0.15914 ADHEr, ATPS4r, FUM, GLCpts, NADH6, THD2 and/orGLUDy 415 6 4.6568 0.25634 ADHEr, ATPS4r, ICL, MDH, PGL and/or G6PDHy,PPS 416 6 4.6568 0.25634 ADHEr, ATPS4r, FUM, MDH, PGL and/or G6PDHy, PPS417 6 4.6568 0.25634 ADHEr, ATPS4r, MALS, MDH, PGL and/or G6PDHy, PPS418 6 4.64861 0.22305 ADHEr, HEX1, LDH_D, PFLi, PPCK, THD2 and/or GLUDy419 6 4.61213 0.23585 ADHEr, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy420 6 4.54731 0.14834 ADHEr, EDA and/or PGDHY, GLUDy, LDH_D, MDH, THD2and/or GLUDy 421 6 4.45035 0.18822 ADHEr, ASPT, ATPS4r, MDH, PYK, RPE422 6 4.44107 0.285 ADHEr, ATPS4r, FUM, HEX1, LDH_D, NADH6 423 6 4.378480.18825 ADHEr, ASPT, ATPS4r, MDH, PYK, TAL 424 6 4.35711 0.22671 ADHEr,ASPT, LDH_D, MDH, NADH12, NADH6 425 6 4.30745 0.05232 ACKr and/or PTAr,ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy 426 6 4.29111 0.05856 ADHEr,ASPT, LDH_D, MDH, PGI, THD2 and/or GLUDy 427 6 4.29026 0.24034 ACKrand/or PTAr, ADHEr, EDA and/or PGDHY, G5SD, GLYCL, PGI 428 6 4.290260.24034 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, GLU5K, GLYCL, PGI 4296 4.19017 0.18892 ADHEr, ATPS4r, FUM, LDH_D, NADH6, THD2 and/or GLUDy430 6 4.14405 0.19951 ADHEr, ATPS4r, FUM, LDH_D, PGDH, PPCK 431 64.14405 0.19951 ADHEr, ATPS4r, FUM, LDH_D, PGL and/or G6PDHy, PPCK 432 64.14405 0.19951 ADHEr, ATPS4r, LDH_D, MDH, PGDH, PPCK 433 6 4.138430.19251 ADHEr, ATPS4r, EDA and/or PGDHY, MDH, PPCK, RPE 434 6 4.118650.20026 ADHEr, ATPS4r, LDH_D, MDH, PPCK, TAL 435 6 4.11865 0.20026ADHEr, ATPS4r, FUM, LDH_D, PPCK, TAL 436 6 4.10258 0.11665 ACKr and/orPTAr, ADHEr, ATPS4r, PPCK, PYK, SUCOAS 437 6 4.09791 0.11017 ADHEr,ATPS4r, LDH_D, MDH, PPCK, THD2 and/or GLUDy 438 6 4.09791 0.11017 ADHEr,ATPS4r, FUM, LDH_D, PPCK, THD2 and/or GLUDy 439 6 4.09533 0.20095 ADHEr,ATPS4r, LDH_D, MDH, PPCK, RPE 440 6 4.09533 0.20095 ADHEr, ATPS4r, FUM,LDH_D, PPCK, RPE 441 6 4.08336 0.13789 ADHEr, ASPT, LDH_D, MDH, PPCK,THD2 and/or GLUDy 442 6 4.08234 0.13828 ADHEr, ASPT, GLCpts, LDH_D, MDH,THD2 and/or GLUDy 443 6 4.0683 0.31169 ADHEr, FUM, HEX1, LDH_D, PPS,THD2 and/or GLUDy 444 6 4.05789 0.14762 ADHEr, ASPT, LDH_D, MDH, NADH6,THD2 and/or GLUDy 445 6 4.04139 0.17129 ADHEr, ASPT, LDH_D, MDH, RPE,THD2 and/or GLUDy 446 6 3.97871 0.17786 ADHEr, ASPT, GLCpts, LDH_D, MDH,PPCK 447 6 3.95205 0.10694 ACKr and/or PTAr, ADHEr, ATPS4r, PGDH, PGI,SUCOAS 448 6 3.95184 0.18812 ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK 449 63.94624 0.19026 ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6 450 6 3.944550.21245 ADHEr, ASPT, LDH_D, MDH, PPCK, RPE 451 6 3.94203 0.1079 ACKrand/or PTAr, ADHEr, ATPS4r, PGI, SUCOAS, TAL 452 6 3.93874 0.25675 ACKrand/or PTAr, ADHEr, FUM, HEX1, LDH_D, THD2 and/or GLUDy 453 6 3.932730.1088 ACKr and/or PTAr, ADHEr, ATPS4r, PGI, RPE, SUCOAS 454 6 3.911860.22634 ADHEr, ASPT, LDH_D, MDH, NADH6, RPE 455 6 3.90038 0.04761 ADHEr,GLCpts, NADH6, PGI, PPCK, THD2 and/or GLUDy 456 6 3.8947 0.05032 ADHEr,FUM, GLCpts, NADH6, PGI, THD2 and/or GLUDy 457 6 3.8947 0.05032 ADHEr,GLCpts, MDH, NADH6, PGI, THD2 and/or GLUDy 458 6 3.84046 0.12564 ACKrand/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, THD2 and/or GLUDy 459 6 3.812280.08972 ADHEr, EDA and/or PGDHY, MDH, NADH6, PYK, THD2 and/or GLUDy 4606 3.81228 0.08972 ADHEr, MDH, NADH6, PGL and/or G6PDHy, PYK, THD2 and/orGLUDy 461 6 3.76251 0.07312 ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PPCK,THD2 and/or GLUDy 462 6 3.70324 0.1395 ACKr and/or PTAr, ADHEr, LDH_D,NADH6, PPCK, PYK 463 6 3.70023 0.1556 ACKr and/or PTAr, ADHEr, LDH_D,MDH, PPCK, PYK 464 6 3.70023 0.1556 ACKr and/or PTAr, ADHEr, FUM, LDH_D,PPCK, PYK 465 6 3.67651 0.1546 ADHEr, MDH, NADH6, PGL and/or G6PDHy,PPCK, PYK 466 6 3.67651 0.1546 ADHEr, EDA and/or PGDHY, MDH, NADH6,PPCK, PYK 467 6 3.652 0.16631 ADHEr, LDH_D, MDH, NADH12, NADH6, THD2and/or GLUDy 468 6 3.64497 0.19156 ADHEr, FUM, GLCpts, NADH6, RPE, THD2and/or GLUDy 469 6 3.62254 0.1804 ADHEr, LDH_D, NADH12, NADH6, PPCK,THD2 and/or GLUDy 470 6 3.5494 0.21535 ADHEr, FUM, LDH_D, NADH12, NADH6,THD2 and/or GLUDy 471 6 3.41696 0.27865 ADHEr, DAAD, FDH2, NADH12,NADH6, PRO1z 472 6 3.41696 0.27865 ADHEr, ALAR, FDH2, NADH12, NADH6,PRO1z 473 6 3.40228 0.13474 ADHEr, ATPS4r, FRD and/or SUCD4, HEX1,LDH_D, THD2 and/or GLUDy 474 6 3.27381 0.03414 ACKr and/or PTAr, ADHEr,FUM, LDH_D, PPCK, THD2 and/or GLUDy 475 6 3.22868 0.19148 ADHEr, FRDand/or SUCD4, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy 476 6 3.119050.12289 ADHEr, FUM, LDH_D, PPCK, PYK, THD2 and/or GLUDy 477 6 3.119050.12289 ADHEr, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy 478 6 3.107770.12936 ADHEr, FUM, GLCpts, LDH_D, PPCK, THD2 and/or GLUDy 479 6 3.107770.12936 ADHEr, GLCpts, LDH_D, MDH, PPCK, THD2 and/or GLUDy 480 6 2.94270.20328 ADHEr, FRD and/or SUCD4, LDH_D, MALS, PPS, THD2 and/or GLUDy 4816 2.9427 0.20328 ADHEr, FRD and/or SUCD4, ICL, LDH_D, PPS, THD2 and/orGLUDy 482 6 2.80361 0.3231 ADHEr, FUM, HEX1, LDH_D, TAL, THD2 and/orGLUDy 483 6 2.79375 0.29126 ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D,THD2 and/or GLUDy 484 6 2.68652 0.29222 ADHEr, ATPS4r, GLUDy, HEX1, RPE,THD2 and/or GLUDy 485 6 2.62059 0.29297 ADHEr, ATPS4r, GLUDy, HEX1, TAL,THD2 and/or GLUDy 486 6 2.46956 0.30762 ADHEr, FUM, LDH_D, PPS, SUCOAS,THD2 and/or GLUDy 487 6 0.97386 0.28207 ACKr and/or PTAr, ADHEr, FUM,MDH, PGL and/or G6PDHy, SUCOAS 488 6 0.97386 0.28207 ACKr and/or PTAr,ADHEr, ICL, MDH, PGL and/or G6PDHy, SUCOAS 489 6 0.97386 0.28207 ACKrand/or PTAr, ADHEr, FUM, MDH, PGDH, SUCOAS 490 6 0.97386 0.28207 ACKrand/or PTAr, ADHEr, MALS, MDH, PGL and/or G6PDHy, SUCOAS 491 6 0.973860.28207 ACKr and/or PTAr, ADHEr, ICL, MDH, PGDH, SUCOAS 492 6 0.973860.28207 ACKr and/or PTAr, ADHEr, MALS, MDH, PGDH, SUCOAS 493 6 0.906860.28301 ACKr and/or PTAr, ADHEr, MALS, MDH, SUCOAS, TAL 494 6 0.906860.28301 ACKr and/or PTAr, ADHEr, ICL, MDH, SUCOAS, TAL 495 6 0.845370.28387 ACKr and/or PTAr, ADHEr, ICL, MDH, RPE, SUCOAS 496 6 0.845370.28387 ACKr and/or PTAr, ADHEr, MALS, MDH, RPE, SUCOAS 497 3 3.262720.35236 ADHEr, LDH_D, NADH6 498 3 2.8403 0.28275 ADHEr, LDH_D, PPCK 4993 1.72604 0.38773 ADHEr, ATPS4r, LDH_D 500 3 0.89333 0.37885 ADHEr, FUM,LDH_D 501 3 0.83466 0.26712 ADHEr, LDH_D, PGI 502 3 0.41621 0.38953ADHEr, HEX1, LDH_D 503 4 5.71646 0.21908 ADHEr, HEX1, LDH_D, PGI 504 44.83364 0.29669 ADHEr, LDH_D, NADH6, PFLi 505 4 4.11897 0.24338 ADHEr,EDA and/or PGDHY, LDH_D, PGI 506 4 3.77533 0.25553 ADHEr, ASPT, LDH_D,MDH 507 4 3.65248 0.19372 ADHEr, LDH_D, NADH6, PGI 508 4 3.47283 0.25194ADHEr, LDH_D, NADH6, PPCK 509 4 3.39319 0.29001 ADHEr, LDH_D, MDH, NADH6510 4 3.35305 0.34906 ADHEr, LDH_D, NADH6, RPE 511 4 3.0877 0.16431ADHEr, LDH_D, PGI, PPCK 512 4 2.90019 0.24841 ADHEr, GLCpts, LDH_D, PPCK513 4 2.89855 0.2815 ADHEr, LDH_D, PPCK, RPE 514 4 2.88617 0.25645ADHEr, FUM, LDH_D, THD2 and/or GLUDy 515 4 2.72186 0.35068 ADHEr, FUM,HEX1, LDH_D 516 4 2.0373 0.38161 ADHEr, ATPS4r, LDH_D, RPE 517 4 1.890070.3845 ADHEr, ATPS4r, LDH_D, TAL 518 4 1.49147 0.26024 ADHEr, LDH_D,PFLi, PGI 519 4 0.5753 0.38695 ADHEr, HEX1, LDH_D, RPE 520 4 0.499680.38818 ADHEr, HEX1, LDH_D, TAL 521 5 6.93034 0.18126 ADHEr, EDA and/orPGDHY, LDH_D, NADH6, PGI 522 5 6.92332 0.16482 ADHEr, HEX1, LDH_D,NADH6, PGI 523 5 6.91845 0.19552 ADHEr, HEX1, LDH_D, PFLi, PGI 524 56.89839 0.18171 ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PGI 525 5 6.148990.1758 ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI 526 5 5.8701 0.13379ADHEr, EDA and/or PGDHY, LDH_D, PGI, PPCK 527 5 5.85757 0.23874 ADHEr,LDH_D, MDH, NADH6, THD2 and/or GLUDy 528 5 5.85411 0.19685 ADHEr, HEX1,LDH_D, PGI, PPS 529 5 5.81422 0.16481 ADHEr, ATPS4r, EDA and/or PGDHY,LDH_D, PGI 530 5 5.40131 0.26362 ADHEr, LDH_D, MDH, PFLi, THD2 and/orGLUDy 531 5 4.88764 0.27849 ADHEr, LDH_D, NADH12, NADH6, PFLi 532 54.88489 0.27942 ADHEr, FUM, LDH_D, NADH6, PFLi 533 5 4.81778 0.26037ADHEr, ATPS4r, LDH_D, MDH, NADH6 534 5 4.73419 0.21859 ADHEr, LDH_D,NADH6, PPCK, THD2 and/or GLUDy 535 5 4.63783 0.29595 ADHEr, ATPS4r,FDH2, LDH_D, NADH6 536 5 4.30547 0.19131 ADHEr, ATPS4r, LDH_D, NADH6,PGI 537 5 4.2733 0.23078 ADHEr, ATPS4r, LDH_D, NADH6, PPCK 538 5 4.238030.24209 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, LDH_D, PGI 539 54.12032 0.32701 ADHEr, ATPS4r, FUM, LDH_D, NADH6 540 5 3.9718 0.23354ADHEr, ATPS4r, LDH_D, MDH, PPCK 541 5 3.9718 0.23354 ADHEr, ATPS4r, FUM,LDH_D, PPCK 542 5 3.8747 0.21758 ADHEr, ASPT, GLCpts, LDH_D, MDH 543 53.84814 0.25342 ADHEr, ASPT, LDH_D, MDH, RPE 544 5 3.83986 0.2047 ADHEr,ASPT, LDH_D, MDH, PYK 545 5 3.75472 0.32987 ADHEr, ATPS4r, HEX1, LDH_D,NADH6 546 5 3.54965 0.29114 ADHEr, ATPS4r, LDH_D, MDH, PGDH 547 53.54605 0.21695 ADHEr, GLCpts, LDH_D, NADH6, PPCK 548 5 3.54385 0.218ADHEr, LDH_D, NADH6, PPCK, PYK 549 5 3.53615 0.25027 ADHEr, LDH_D,NADH6, PPCK, RPE 550 5 3.5018 0.32809 ADHEr, ATPS4r, FUM, HEX1, LDH_D551 5 3.46904 0.25375 ADHEr, GLCpts, LDH_D, MDH, NADH6 552 5 3.465280.28851 ADHEr, LDH_D, MDH, NADH6, RPE 553 5 3.44916 0.13425 ADHEr,LDH_D, PFLi, PGI, PPCK 554 5 3.44555 0.26498 ADHEr, HEX1, LDH_D, NADH6,THD2 and/or GLUDy 555 5 3.43776 0.29402 ADHEr, ATPS4r, LDH_D, MDH, TAL556 5 3.33377 0.2967 ADHEr, ATPS4r, LDH_D, MDH, RPE 557 5 3.331520.33978 ADHEr, FUM, LDH_D, NADH6, TAL 558 5 3.32935 0.34088 ADHEr, HEX1,LDH_D, NADH6, TAL 559 5 3.32788 0.32122 ADHEr, FUM, HEX1, LDH_D, NADH6560 5 3.31278 0.3493 ADHEr, G5SD, LDH_D, NADH6, TAL 561 5 3.31278 0.3493ADHEr, GLU5K, LDH_D, NADH6, TAL 562 5 3.23462 0.28717 ACKr and/or PTAr,ADHEr, ATPS4r, LDH_D, SUCOAS 563 5 3.17484 0.10602 ADHEr, LDH_D, PGI,PPCK, THD2 and/or GLUDy 564 5 3.16614 0.11184 ADHEr, LDH_D, MDH, PGI,THD2 and/or GLUDy 565 5 3.16614 0.11184 ADHEr, FUM, LDH_D, PGI, THD2and/or GLUDy 566 5 3.11125 0.24826 ADHEr, ATPS4r, LDH_D, PPCK, THD2and/or GLUDy 567 5 2.95529 0.24477 ADHEr, LDH_D, PPCK, RPE, THD2 and/orGLUDy 568 5 2.95136 0.24731 ADHEr, GLCpts, LDH_D, PPCK, RPE 569 52.94249 0.25305 ADHEr, FUM, LDH_D, RPE, THD2 and/or GLUDy 570 5 2.937650.22693 ADHEr, FUM, LDH_D, PPCK, PYK 571 5 2.93765 0.22693 ADHEr, LDH_D,MDH, PPCK, PYK 572 5 2.9332 0.24406 ADHEr, LDH_D, PPCK, TAL, THD2 and/orGLUDy 573 5 2.90913 0.24328 ADHEr, LDH_D, PGDH, PPCK, THD2 and/or GLUDy574 5 2.90913 0.24328 ADHEr, LDH_D, PGL and/or G6PDHy, PPCK, THD2 and/orGLUDy 575 5 2.90081 0.26381 ADHEr, LDH_D, MDH, PPCK, TAL 576 5 2.900810.26381 ADHEr, FUM, LDH_D, PPCK, TAL 577 5 2.79498 0.34856 ADHEr, FUM,HEX1, LDH_D, RPE 578 5 2.61943 0.36027 ADHEr, HEX1, LDH_D, PFLi, PPS 5795 2.60615 0.3202 ADHEr, ATPS4r, HEX1, LDH_D, PFLi 580 5 2.60314 0.31113ADHEr, HEX1, LDH_D, MDH, PFLi 581 5 2.54001 0.22798 ADHEr, LDH_D, PFLi,PGDH, PGI 582 5 2.5259 0.22921 ADHEr, LDH_D, PFLi, PGI, TAL 583 5 2.51290.23034 ADHEr, LDH_D, PFLi, PGI, RPE 584 5 2.50442 0.16853 ADHEr,ATPS4r, LDH_D, PFLi, PGI 585 5 2.45433 0.3546 ADHEr, ATPS4r, HEX1,LDH_D, THD2 and/or GLUDy 586 5 2.18633 0.3609 ADHEr, HEX1, LDH_D, PPS,THD2 and/or GLUDy 587 5 2.11347 0.31979 ADHEr, ATPS4r, HEX1, LDH_D, MDH588 5 1.5933 0.25891 ADHEr, FUM, LDH_D, PFLi, PGI 589 5 0.9475 0.3633ADHEr, FUM, LDH_D, PGDH, TAL 590 5 0.9475 0.3633 ADHEr, FUM, LDH_D, PGLand/or G6PDHy, TAL 591 6 7.08404 0.12641 ADHEr, EDA and/or PGDHY, LDH_D,NADH6, PFLi, PGI 592 6 7.0245 0.10838 ADHEr, EDA and/or PGDHY, LDH_D,PFLi, PGI, PPCK 593 6 6.98742 0.06748 ADHEr, EDA and/or PGDHY, LDH_D,NADH6, PGI, THD2 and/or GLUDy 594 6 6.69134 0.13239 ADHEr, ASPT, ATPS4r,GLCpts, LDH_D, MDH 595 6 6.58534 0.16513 ADHEr, EDA and/or PGDHY,GLCpts, LDH_D, PFLi, PGI 596 6 6.55123 0.09841 ADHEr, ASPT, LDH_D, MDH,PGL and/or G6PDHy, PYK 597 6 6.55123 0.09841 ADHEr, ASPT, EDA and/orPGDHY, LDH_D, MDH, PYK 598 6 6.36357 0.11937 ADHEr, EDA and/or PGDHY,LDH_D, PFLi, PGI, THD2 and/or GLUDy 599 6 6.22082 0.11375 ADHEr, ATPS4r,EDA and/or PGDHY, LDH_D, PFLi, PGI 600 6 5.98165 0.07186 ADHEr, ATPS4r,EDA and/or PGDHY, LDH_D, PGI, PPCK 601 6 5.93527 0.09761 ADHEr, EDAand/or PGDHY, GLCpts, LDH_D, PGI, PPCK 602 6 5.87444 0.0434 ADHEr, EDAand/or PGDHY, FUM, LDH_D, PGI, THD2 and/or GLUDy 603 6 5.87444 0.0434ADHEr, EDA and/or PGDHY, LDH_D, MDH, PGI, THD2 and/or GLUDy 604 65.65488 0.26204 ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFLi, THD2 and/orGLUDy 605 6 5.59555 0.20952 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6 6066 5.1776 0.13724 ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI 607 6 5.117440.13758 ADHEr, FUM, LDH_D, NADH6, PFLi, PGI 608 6 5.11744 0.13758 ADHEr,LDH_D, MDH, NADH6, PFLi, PGI 609 6 5.09877 0.17589 ADHEr, ATPS4r,GLCpts, LDH_D, NADH6, PPCK 610 6 5.08219 0.13778 ADHEr, ATPS4r, LDH_D,NADH6, PFLi, PGI 611 6 5.02693 0.21085 ADHEr, ATPS4r, LDH_D, NADH6,PGDH, PPCK 612 6 5.02693 0.21085 ADHEr, ATPS4r, LDH_D, NADH6, PGL and/orG6PDHy, PPCK 613 6 5.00057 0.21154 ADHEr, ATPS4r, LDH_D, NADH6, PPCK,TAL 614 6 4.97638 0.21218 ADHEr, ATPS4r, LDH_D, NADH6, PPCK, RPE 615 64.88512 0.29281 ADHEr, GLU5K, LDH_D, NADH6, PFLi, RPE 616 6 4.885120.29281 ADHEr, G5SD, LDH_D, NADH6, PFLi, RPE 617 6 4.885 0.29286 ADHEr,ASNS2, LDH_D, NADH6, PFLi, RPE 618 6 4.85776 0.29446 ADHEr, ATPS4r,FDH2, LDH_D, NADH6, RPE 619 6 4.83644 0.29517 ADHEr, ATPS4r, FDH2,LDH_D, NADH6, TAL 620 6 4.66386 0.18074 ADHEr, ATPS4r, FUM, GLCpts,LDH_D, PPCK 621 6 4.66386 0.18074 ADHEr, ATPS4r, GLCpts, LDH_D, MDH,PPCK 622 6 4.63095 0.20189 ADHEr, ATPS4r, GLCpts, LDH_D, PFLi, PPCK 6236 4.49707 0.25782 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, THD2and/or GLUDy 624 6 4.4958 0.1778 ADHEr, ASPT, LDH_D, MDH, NADH6, PYK 6256 4.41977 0.25646 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, THD2 and/or GLUDy626 6 4.18509 0.32432 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGL and/orG6PDHy 627 6 4.18509 0.32432 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PGDH 6286 4.15013 0.1124 ADHEr, ASPT, LDH_D, MDH, PYK, THD2 and/or GLUDy 629 64.14582 0.32483 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, TAL 630 6 4.10990.32529 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, RPE 631 6 4.04473 0.12883ADHEr, LDH_D, PFLi, PGDH, PGI, PPCK 632 6 4.03844 0.12934 ADHEr, LDH_D,PFLi, PGI, PPCK, TAL 633 6 4.03266 0.12981 ADHEr, LDH_D, PFLi, PGI,PPCK, RPE 634 6 4.02531 0.1111 ADHEr, GLCpts, LDH_D, PFLi, PGI, PPCK 6356 3.9367 0.21579 ADHEr, ASPT, GLCpts, LDH_D, MDH, RPE 636 6 3.760490.30843 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi 637 6 3.71345 0.27754ADHEr, ATPS4r, LDH_D, MDH, PGDH, TAL 638 6 3.69299 0.14673 ADHEr, LDH_D,NADH6, PPCK, PYK, THD2 and/or GLUDy 639 6 3.64625 0.1409 ACKr and/orPTAr, ADHEr, LDH_D, MDH, PGI, PPCK 640 6 3.64625 0.1409 ACKr and/orPTAr, ADHEr, FUM, LDH_D, PGI, PPCK 641 6 3.60057 0.21551 ADHEr, GLCpts,LDH_D, NADH6, PPCK, RPE 642 6 3.59735 0.21725 ADHEr, LDH_D, NADH6, PPCK,PYK, RPE 643 6 3.59622 0.19297 ADHEr, FUM, GLCpts, LDH_D, NADH6, PPCK644 6 3.59622 0.19297 ADHEr, GLCpts, LDH_D, MDH, NADH6, PPCK 645 63.59594 0.19311 ADHEr, FUM, LDH_D, NADH6, PPCK, PYK 646 6 3.595940.19311 ADHEr, LDH_D, MDH, NADH6, PPCK, PYK 647 6 3.59157 0.1952 ADHEr,FUM, GLCpts, LDH_D, NADH6, THD2 and/or GLUDy 648 6 3.55204 0.22769ADHEr, LDH_D, MDH, NADH6, PPCK, TAL 649 6 3.55204 0.22769 ADHEr, FUM,LDH_D, NADH6, PPCK, TAL 650 6 3.53212 0.25244 ADHEr, GLCpts, LDH_D, MDH,NADH6, RPE 651 6 3.52326 0.22785 ADHEr, GLU5K, LDH_D, MDH, NADH6, PPCK652 6 3.52326 0.22785 ADHEr, G5SD, LDH_D, MDH, NADH6, PPCK 653 6 3.523260.22785 ADHEr, FUM, GLU5K, LDH_D, NADH6, PPCK 654 6 3.52326 0.22785ADHEr, FUM, G5SD, LDH_D, NADH6, PPCK 655 6 3.52319 0.22788 ADHEr, ASNS2,FUM, LDH_D, NADH6, PPCK 656 6 3.52319 0.22788 ADHEr, ASNS2, LDH_D, MDH,NADH6, PPCK 657 6 3.51328 0.26261 ADHEr, HEX1, LDH_D, NADH6, RPE, THD2and/or GLUDy 658 6 3.40958 0.31856 ADHEr, FUM, HEX1, LDH_D, NADH6, RPE659 6 3.19551 0.34972 ADHEr, ATPS4r, HEX1, LDH_D, PPS, THD2 and/or GLUDy660 6 2.95797 0.22895 ADHEr, FUM, GLCpts, LDH_D, PPCK, TAL 661 6 2.957970.22895 ADHEr, GLCpts, LDH_D, MDH, PPCK, TAL 662 6 2.94768 0.30664ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi 663 6 2.92149 0.26881 ADHEr,ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPS 664 6 2.76831 0.35787 ADHEr,HEX1, LDH_D, PFLi, PPS, RPE 665 6 2.7362 0.31504 ADHEr, ATPS4r, HEX1,LDH_D, PFLi, RPE 666 6 2.73201 0.30945 ADHEr, HEX1, LDH_D, MDH, PFLi,RPE 667 6 2.69754 0.35901 ADHEr, HEX1, LDH_D, PFLi, PPS, TAL 668 62.67469 0.31748 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, TAL 669 6 2.670710.31025 ADHEr, HEX1, LDH_D, MDH, PFLi, TAL 670 6 2.61965 0.35455 ADHEr,ATPS4r, HEX1, LDH_D, RPE, THD2 and/or GLUDy 671 6 2.60629 0.2178 ADHEr,LDH_D, PFLi, PGDH, PGI, TAL 672 6 2.5479 0.2938 ADHEr, ATPS4r, GLUDy,HEX1, LDH_D, THD2 and/or GLUDy 673 6 2.5408 0.35457 ADHEr, ATPS4r, HEX1,LDH_D, TAL, THD2 and/or GLUDy 674 6 2.05497 0.19746 ADHEr, ASPT, ATPS4r,LDH_D, PGI, THD5 675 6 1.65594 0.25767 ACKr and/or PTAr, ADHEr, FUM,LDH_D, PFLi, PGI 676 7 7.90077 0.01226 ADHEr, ASPT, LDH_D, MDH, PFLi,PPCK, THD2 and/or GLUDy 677 7 7.84104 0.01965 ADHEr, ASPT, LDH_D, MDH,NADH6, PFLi, THD2 and/or GLUDy 678 7 7.7052 0.03644 ADHEr, ASPT, HEX1,LDH_D, MDH, PFLi, THD2 and/or GLUDy 679 7 7.67191 0.04055 ADHEr, ASPT,GLCpts, LDH_D, MDH, PFLi, THD2 and/or GLUDy 680 7 7.61518 0.06956 ADHEr,ATPS4r, LDH_D, MDH, NADH12, PFLi, THD2 and/or GLUDy 681 7 7.579420.07603 ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, PGDH 682 7 7.579420.07603 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PGDH 683 7 7.576060.07664 ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, TAL 684 7 7.576060.07664 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, TAL 685 7 7.57295 0.0772ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, RPE 686 7 7.57295 0.0772 ADHEr,ATPS4r, LDH_D, MDH, NADH6, PFLi, RPE 687 7 7.55163 0.08105 ADHEr, LDH_D,MDH, NADH12, NADH6, PFLi, THD2 and/or GLUDy 688 7 7.38115 0.07649 ADHEr,ASPT, ATPS4r, GLCpts, LDH_D, MDH, PFLi 689 7 7.293 0.07152 ADHEr, ASPT,ATPS4r, GLCpts, LDH_D, MDH, NADH6 690 7 7.21161 0.05631 ADHEr, EDAand/or PGDHY, HEX1, LDH_D, NADH6, PGI, THD2 and/or GLUDy 691 7 7.109530.03056 ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PYK, THD2 and/orGLUDy 692 7 7.04629 0.01861 ADHEr, ARGt4, ASPT, LDH_D, MALS, MDH, THD2and/or GLUDy 693 7 7.04629 0.01861 ADHEr, ARGt4, ASPT, ICL, LDH_D, MDH,THD2 and/or GLUDy 694 7 7.01619 0.17329 ACKr and/or PTAr, ADHEr, ATPS4r,LDH_D, MDH, NADH6, PGDH 695 7 7.00623 0.10284 ADHEr, LDH_D, MDH, NADH12,NADH6, PFLi, PGI 696 7 7.00542 0.17519 ACKr and/or PTAr, ADHEr, ATPS4r,LDH_D, MDH, NADH6, TAL 697 7 6.99744 0.05155 ACKr and/or PTAr, ADHEr,ICL, LDH_D, MDH, NADH6, THD2 and/or GLUDy 698 7 6.99744 0.05155 ACKrand/or PTAr, ADHEr, FUM, LDH_D, MDH, NADH6, THD2 and/or GLUDy 699 76.99744 0.05155 ACKr and/or PTAr, ADHEr, LDH_D, MALS, MDH, NADH6, THD2and/or GLUDy 700 7 6.99618 0.02608 ADHEr, ASPT, ATPS4r, GLCpts, LDH_D,MDH, THD2 and/or GLUDy 701 7 6.99539 0.17696 ACKr and/or PTAr, ADHEr,ATPS4r, LDH_D, MDH, NADH6, RPE 702 7 6.99098 0.13845 ADHEr, ASPT,ATPS4r, LDH_D, MDH, PFLi, PGL and/or G6PDHy 703 7 6.99098 0.13845 ADHEr,ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, PFLi 704 7 6.97749 0.0848ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGI 705 7 6.954950.04834 ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, NADH6, THD2 and/orGLUDy 706 7 6.94643 0.04549 ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK, THD2and/or GLUDy 707 7 6.94643 0.04549 ADHEr, ATPS4r, FUM, LDH_D, PFLi,PPCK, THD2 and/or GLUDy 708 7 6.94035 0.16409 ACKr and/or PTAr, ADHEr,ASPT, ATPS4r, LDH_D, MDH, PGDH 709 7 6.93216 0.19304 ADHEr, HEX1, LDH_D,MDH, PFLi, PPS, THD2 and/or GLUDy 710 7 6.9218 0.16465 ACKr and/or PTAr,ADHEr, ASPT, ATPS4r, LDH_D, MDH, TAL 711 7 6.90477 0.16517 ACKr and/orPTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, RPE 712 7 6.89223 0.07039 ADHEr,LDH_D, NADH12, NADH6, PFLi, PPCK, THD2 and/or GLUDy 713 7 6.890880.16538 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, PGDH 714 76.88301 0.07117 ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK, THD2 and/or GLUDy715 7 6.88301 0.07117 ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK, THD2 and/orGLUDy 716 7 6.86164 0.16622 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY,LDH_D, MDH, TAL 717 7 6.83471 0.167 ADHEr, ASPT, ATPS4r, EDA and/orPGDHY, LDH_D, MDH, RPE 718 7 6.81823 0.05671 ADHEr, ATPS4r, LDH_D,NADH12, PFLi, PPCK, THD2 and/or GLUDy 719 7 6.75858 0.12153 ADHEr,GLCpts, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy 720 7 6.75858 0.12153ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK, THD2 and/or GLUDy 721 7 6.743540.0559 ADHEr, GLUDy, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy 722 76.72704 0.08076 ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, NADH6, PYK723 7 6.72704 0.08076 ADHEr, ASPT, LDH_D, MDH, NADH6, PGL and/or G6PDHy,PYK 724 7 6.72478 0.09245 ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D,PPCK, THD2 and/or GLUDy 725 7 6.71779 0.1313 ADHEr, FBP, LDH_D, MDH,PFLi, PGDH, THD2 and/or GLUDy 726 7 6.71371 0.1397 ADHEr, GLCpts, LDH_D,MDH, NADH6, PFLi, THD2 and/or GLUDy 727 7 6.69882 0.08259 ADHEr, ATPS4r,LDH_D, MDH, PFLi, PGDH, THD2 and/or GLUDy 728 7 6.69814 0.08084 ADHEr,LDH_D, MDH, NADH6, PFLi, PGDH, THD2 and/or GLUDy 729 7 6.68517 0.13281ADHEr, FBP, LDH_D, MDH, PFLi, TAL, THD2 and/or GLUDy 730 7 6.678270.0833 ADHEr, ATPS4r, LDH_D, MDH, PFLi, TAL, THD2 and/or GLUDy 731 76.67803 0.08153 ADHEr, LDH_D, MDH, NADH6, PFLi, TAL, THD2 and/or GLUDy732 7 6.66553 0.13149 ADHEr, LDH_D, MDH, PFLi, PGDH, PGI, THD2 and/orGLUDy 733 7 6.65922 0.08397 ADHEr, ATPS4r, LDH_D, MDH, PFLi, RPE, THD2and/or GLUDy 734 7 6.65477 0.13422 ADHEr, FBP, LDH_D, MDH, PFLi, RPE,THD2 and/or GLUDy 735 7 6.65444 0.13292 ADHEr, LDH_D, MDH, PFLi, PGI,TAL, THD2 and/or GLUDy 736 7 6.64411 0.13426 ADHEr, LDH_D, MDH, PFLi,PGI, RPE, THD2 and/or GLUDy 737 7 6.54706 0.05865 ADHEr, ATPS4r, EDAand/or PGDHY, LDH_D, MDH, NADH6, RPE 738 7 6.50546 0.0488 ADHEr, ASPT,EDA and/or PGDHY, FUM, LDH_D, MDH, THD2 and/or GLUDy 739 7 6.49510.09302 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, RPE, THD2 and/orGLUDy 740 7 6.48152 0.09261 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D,PPCK, TAL, THD2 and/or GLUDy 741 7 6.4815 0.14937 ACKr and/or PTAr,ADHEr, LDH_D, MDH, NADH6, RPE, THD2 and/or GLUDy 742 7 6.4814 0.1488ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, TAL, THD2 and/or GLUDy 743 76.48129 0.14819 ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PGDH, THD2and/or GLUDy 744 7 6.46677 0.09217 ACKr and/or PTAr, ADHEr, ATPS4r,LDH_D, PGDH, PPCK, THD2 and/or GLUDy 745 7 6.46677 0.09217 ACKr and/orPTAr, ADHEr, ATPS4r, LDH_D, PGL and/or G6PDHy, PPCK, THD2 and/or GLUDy746 7 6.40404 0.13985 ACKr and/or PTAr, ADHEr, ASPT, GLCpts, LDH_D, MDH,NADH6 747 7 6.39358 0.0544 ADHEr, ASPT, FUM, GLCpts, LDH_D, MDH, THD2and/or GLUDy 748 7 6.36333 0.16503 ACKr and/or PTAr, ADHEr, ASPT, LDH_D,MDH, NADH6, THD2 and/or GLUDy 749 7 6.35537 0.14906 ACKr and/or PTAr,ADHEr, ASPT, LDH_D, MDH, NADH6, PYK 750 7 6.34075 0.15004 ACKr and/orPTAr, ADHEr, FDH2, GLCpts, LDH_D, MDH, NADH6 751 7 6.33847 0.16728 ACKrand/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, NADH6 752 7 6.301360.19463 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, THD2 and/orGLUDy 753 7 6.30082 0.1583 ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D,MDH, PYK 754 7 6.23552 0.18763 ACKr and/or PTAr, ADHEr, FDH2, LDH_D,MDH, NADH6, THD2 and/or GLUDy 755 7 6.23519 0.15548 ACKr and/or PTAr,ADHEr, HEX1, LDH_D, MDH, PGDH, THD2 and/or GLUDy 756 7 6.22748 0.15693ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH, TAL, THD2 and/or GLUDy 757 76.22033 0.15828 ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH, RPE, THD2and/or GLUDy 758 7 6.2176 0.1588 ACKr and/or PTAr, ADHEr, ASPT, LDH_D,MDH, PGDH, THD2 and/or GLUDy 759 7 6.21321 0.15963 ACKr and/or PTAr,ADHEr, ASPT, LDH_D, MDH, TAL, THD2 and/or GLUDy 760 7 6.20917 0.16039ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, RPE, THD2 and/or GLUDy 761 76.20871 0.17434 ADHEr, EDA and/or PGDHY, LDH_D, MDH, PFLi, PGI, THD5 7627 6.18575 0.17904 ACKr and/or PTAr, ADHEr, FDH2, LDH_D, MDH, NADH12,NADH6 763 7 6.17771 0.01562 ADHEr, ASPT, ICL, LDH_D, MDH, NADH6, THD2and/or GLUDy 764 7 6.17771 0.01562 ADHEr, ASPT, LDH_D, MALS, MDH, NADH6,THD2 and/or GLUDy 765 7 6.10255 0.12296 ADHEr, ASPT, ATPS4r, LDH_D, MDH,NADH6, PPCK 766 7 6.08364 0.10963 ADHEr, GLCpts, LDH_D, NADH6, PFLi,PPCK, THD2 and/or GLUDy 767 7 6.05909 0.217 ACKr and/or PTAr, ADHEr,ATPS4r, FUM, HEX1, LDH_D, THD2 and/or GLUDy 768 7 5.99909 0.09659 ADHEr,FUM, GLCpts, LDH_D, PFLi, PGI, PPCK 769 7 5.99909 0.09659 ADHEr, GLCpts,LDH_D, MDH, PFLi, PGI, PPCK 770 7 5.9797 0.19373 ACKr and/or PTAr, ACS,ADHEr, HEX1, LDH_D, PGI, PPS 771 7 5.97334 0.11266 ADHEr, GLCpts, LDH_D,MDH, NADH6, PFLi, PGI 772 7 5.94122 0.14646 ACKr and/or PTAr, ADHEr,LDH_D, MDH, PFLi, PPCK, PYK 773 7 5.94122 0.14646 ACKr and/or PTAr,ADHEr, FUM, LDH_D, PFLi, PPCK, PYK 774 7 5.9395 0.02821 ADHEr, ATPS4r,FDH2, LDH_D, MDH, NADH6, THD2 and/or GLUDy 775 7 5.89777 0.06803 ADHEr,ATPS4r, EDA and/or PGDHY, HEX1, LDH_D, PGI, THD2 and/or GLUDy 776 75.86803 0.06511 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPCK, THD2 and/orGLUDy 777 7 5.84763 0.01509 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1,LDH_D, PGI, THD2 and/or GLUDy 778 7 5.80727 0.03994 ADHEr, ATPS4r,GLCpts, LDH_D, MDH, NADH6, THD2 and/or GLUDy 779 7 5.80665 0.23182 ACKrand/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, RPE, THD2 and/or GLUDy 780 75.77313 0.13132 ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, PGI 781 75.77052 0.23124 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, TAL, THD2and/or GLUDy 782 7 5.75832 0.26044 ACKr and/or PTAr, ADHEr, G5SD, LDH_D,MDH, PFLi, THD2 and/or GLUDy 783 7 5.75832 0.26044 ACKr and/or PTAr,ADHEr, GLU5K, LDH_D, MDH, PFLi, THD2 and/or GLUDy 784 7 5.75556 0.13165ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PGI 785 7 5.75413 0.21628 ACKrand/or PTAr, ADHEr, FUM, HEX1, LDH_D, NADH6, THD2 and/or GLUDy 786 75.75142 0.15 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PFLi, PPCK 787 7 5.751420.15 ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PFLi, PPCK 788 7 5.73569 0.11619ACKr and/or PTAr, ADHEr, FUM, LDH_D, NADH6, PPCK, THD2 and/or GLUDy 7897 5.73112 0.23062 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGDH,THD2 and/or GLUDy 790 7 5.73112 0.23062 ACKr and/or PTAr, ADHEr, ATPS4r,HEX1, LDH_D, PGL and/or G6PDHy, THD2 and/or GLUDy 791 7 5.7037 0.1229ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6, PPCK 792 7 5.7037 0.1229ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PPCK 793 7 5.67082 0.04313ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGI, THD2 and/or GLUDy 794 7 5.670820.04313 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGI, THD2 and/or GLUDy 795 75.6689 0.01529 ADHEr, LDH_D, MDH, NADH6, PFLi, PYK, THD2 and/or GLUDy796 7 5.57706 0.11542 ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PFLi, THD2and/or GLUDy 797 7 5.57479 0.20482 ACKr and/or PTAr, ADHEr, ATPS4r,HEX1, LDH_D, MDH, THD2 and/or GLUDy 798 7 5.56128 0.20483 ACKr and/orPTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH12, THD2 and/or GLUDy 799 7 5.536870.15179 ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK, THD2 and/or GLUDy 800 75.48032 0.07882 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, THD2 and/orGLUDy 801 7 5.48032 0.07882 ADHEr, ATPS4r, FDH2, FUM, LDH_D, NADH6, THD2and/or GLUDy 802 7 5.4574 0.07896 ADHEr, ASPT, ATPS4r, LDH_D, MDH, PPCK,THD2 and/or GLUDy 803 7 5.43688 0.14109 ADHEr, ASPT, FUM, HEX1, LDH_D,PPS, THD2 and/or GLUDy 804 7 5.42926 0.03599 ACKr and/or PTAr, ADHEr,GLUDy, LDH_D, MDH, PPCK, THD2 and/or GLUDy 805 7 5.41288 0.10644 ADHEr,FUM, LDH_D, PFLi, PGI, PPCK, RPE 806 7 5.41288 0.10644 ADHEr, LDH_D,MDH, PFLi, PGI, PPCK, RPE 807 7 5.40727 0.10598 ADHEr, FUM, LDH_D, PFLi,PGI, PPCK, TAL 808 7 5.40727 0.10598 ADHEr, LDH_D, MDH, PFLi, PGI, PPCK,TAL 809 7 5.40541 0.10406 ADHEr, LDH_D, NADH12, NADH6, PFLi, PPCK, PYK810 7 5.40329 0.10477 ADHEr, LDH_D, MDH, NADH6, PFLi, PPCK, PYK 811 75.40329 0.10477 ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK, PYK 812 7 5.401170.10549 ADHEr, LDH_D, MDH, PFLi, PGDH, PGI, PPCK 813 7 5.40117 0.10549ADHEr, FUM, LDH_D, PFLi, PGDH, PGI, PPCK 814 7 5.38947 0.11011 ACKrand/or PTAr, ADHEr, FUM, LDH_D, NADH6, PPCK, PYK 815 7 5.37417 0.11441ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PPCK, THD2 and/or GLUDy 8167 5.37043 0.12143 ADHEr, LDH_D, MDH, NADH6, PFLi, PGI, RPE 817 7 5.370150.12013 ADHEr, ASPT, EDA and/or PGDHY, GLUDy, LDH_D, MDH, THD2 and/orGLUDy 818 7 5.36414 0.12087 ADHEr, LDH_D, MDH, NADH6, PFLi, PGI, TAL 8197 5.3573 0.12027 ADHEr, LDH_D, MDH, NADH6, PFLi, PGDH, PGI 820 7 5.35120.12232 ADHEr, LDH_D, NADH12, NADH6, PFLi, PRO1z, THD2 and/or GLUDy 8217 5.35114 0.12824 ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI, RPE 822 75.34992 0.12867 ADHEr, FUM, LDH_D, NADH6, PFLi, PGI, RPE 823 7 5.344210.12775 ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI, TAL 824 7 5.342980.12817 ADHEr, FUM, LDH_D, NADH6, PFLi, PGI, TAL 825 7 5.33668 0.12721ADHEr, LDH_D, NADH12, NADH6, PFLi, PGDH, PGI 826 7 5.33544 0.12763ADHEr, FUM, LDH_D, NADH6, PFLi, PGDH, PGI 827 7 5.32853 0.04869 ADHEr,ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPCK, THD2 and/or GLUDy 828 75.32586 0.13717 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, RPE 829 75.31986 0.13616 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, TAL 830 75.31768 0.14006 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, RPE 831 75.31768 0.14006 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, RPE 832 75.31338 0.13506 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGL and/or G6PDHy,PPCK 833 7 5.31338 0.13506 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGDH, PPCK834 7 5.31161 0.139 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, TAL 835 75.31161 0.139 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, TAL 836 7 5.305070.13786 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGL and/or G6PDHy, PPCK 837 75.30507 0.13786 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH, PPCK 838 75.30507 0.13786 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGDH, PPCK 839 75.30121 0.14588 ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK, RPE 840 75.29298 0.14544 ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK, TAL 841 75.29144 0.14245 ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, PFLi 842 75.28753 0.08933 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PPCK, THD2 and/orGLUDy 843 7 5.28422 0.14489 ADHEr, ATPS4r, FDH2, LDH_D, NADH12, NADH6,THD2 and/or GLUDy 844 7 5.28422 0.14489 ADHEr, ATPS4r, LDH_D, NADH12,NADH6, PFLi, THD2 and/or GLUDy 845 7 5.28401 0.14496 ADHEr, ATPS4r,GLCpts, LDH_D, NADH6, PGDH, PPCK 846 7 5.28401 0.14496 ADHEr, ATPS4r,GLCpts, LDH_D, NADH6, PGL and/or G6PDHy, PPCK 847 7 5.27588 0.18393ADHEr, FUM, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy 848 7 5.275880.18393 ADHEr, FUM, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/orGLUDy 849 7 5.2736 0.14846 ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi, THD2and/or GLUDy 850 7 5.25759 0.18586 ADHEr, FUM, HEX1, LDH_D, PPS, TAL,THD2 and/or GLUDy 851 7 5.24329 0.06953 ADHEr, ATPS4r, LDH_D, MDH,NADH6, PGDH, THD2 and/or GLUDy 852 7 5.24057 0.18767 ADHEr, FUM, HEX1,LDH_D, PPS, RPE, THD2 and/or GLUDy 853 7 5.23194 0.07004 ADHEr, ATPS4r,LDH_D, MDH, NADH6, TAL, THD2 and/or GLUDy 854 7 5.22143 0.07051 ADHEr,ATPS4r, LDH_D, MDH, NADH6, RPE, THD2 and/or GLUDy 855 7 5.1096 0.03265ADHEr, ASPT, FUM, GLUDy, HEX1, LDH_D, THD2 and/or GLUDy 856 7 5.078330.23187 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, NADH6, THD2 and/orGLUDy 857 7 4.9126 0.20186 ADHEr, HEX1, LDH_D, PGDH, PPS, TAL, THD2and/or GLUDy 858 7 4.9126 0.20186 ADHEr, HEX1, LDH_D, PGL and/or G6PDHy,PPS, TAL, THD2 and/or GLUDy 859 7 4.89537 0.08136 ACKr and/or PTAr,ADHEr, ATPS4r, GLUDy, LDH_D, MDH, THD2 and/or GLUDy 860 7 4.864690.20078 ADHEr, ATPS4r, GLCpts, LDH_D, PFLi, PPCK, RPE 861 7 4.861880.1035 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, PYK, THD2 and/orGLUDy 862 7 4.85917 0.04186 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PPCK,THD2 and/or GLUDy 863 7 4.85917 0.04186 ADHEr, ATPS4r, FUM, GLCpts,LDH_D, PPCK, THD2 and/or GLUDy 864 7 4.84043 0.16101 ADHEr, ATPS4r,FDH2, LDH_D, NADH6, PPCK, PYK 865 7 4.75352 0.20131 ADHEr, ATPS4r,GLCpts, LDH_D, PFLi, PPCK, TAL 866 7 4.6646 0.15914 ADHEr, ATPS4r, FUM,GLCpts, LDH_D, NADH6, THD2 and/or GLUDy 867 7 4.6568 0.25634 ADHEr,ATPS4r, LDH_D, MALS, MDH, PGL and/or G6PDHy, PPS 868 7 4.6568 0.25634ADHEr, ATPS4r, ICL, LDH_D, MDH, PGL and/or G6PDHy, PPS 869 7 4.65680.25634 ADHEr, ATPS4r, FUM, LDH_D, MDH, PGL and/or G6PDHy, PPS 870 74.60984 0.1687 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, RPE, THD2and/or GLUDy 871 7 4.60771 0.13834 ADHEr, ASPT, LDH_D, MDH, NADH12,NADH6, THD2 and/or GLUDy 872 7 4.5647 0.0741 ADHEr, ATPS4r, HEX1, LDH_D,MDH, SUCOAS, THD2 and/or GLUDy 873 7 4.51839 0.16783 ACKr and/or PTAr,ADHEr, ATPS4r, LDH_D, MDH, TAL, THD2 and/or GLUDy 874 7 4.48284 0.18237ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6, PPCK 875 7 4.46608 0.18828ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH12, NADH6 876 7 4.43524 0.31394ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGDH, PPS 877 7 4.43524 0.31394 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PGL and/or G6PDHy, PPS 878 7 4.43072 0.22439ADHEr, ASPT, LDH_D, MDH, NADH12, NADH6, RPE 879 7 4.41919 0.16689 ACKrand/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PGDH, THD2 and/or GLUDy 880 74.4053 0.01495 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, PPCK, THD2and/or GLUDy 881 7 4.39394 0.31457 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PPS,TAL 882 7 4.37728 0.02565 ADHEr, ASPT, LDH_D, MDH, PGI, PPCK, THD2and/or GLUDy 883 7 4.37061 0.04989 ADHEr, ATPS4r, LDH_D, NADH6, PGI,PPCK, THD2 and/or GLUDy 884 7 4.36653 0.05265 ADHEr, ATPS4r, FUM, LDH_D,NADH6, PGI, THD2 and/or GLUDy 885 7 4.36374 0.03082 ADHEr, ASPT, LDH_D,MDH, NADH6, PGI, THD2 and/or GLUDy 886 7 4.36277 0.03119 ADHEr, ASPT,GLCpts, LDH_D, MDH, PGI, THD2 and/or GLUDy 887 7 4.35613 0.31514 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PPS, RPE 888 7 4.29026 0.24034 ACKr and/orPTAr, ADHEr, EDA and/or PGDHY, GLU5K, GLYCL, LDH_D, PGI 889 7 4.290260.24034 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, G5SD, GLYCL, LDH_D,PGI 890 7 4.25283 0.07318 ADHEr, ASPT, LDH_D, MDH, PPCK, PYK, THD2and/or GLUDy 891 7 4.17894 0.31889 ADHEr, HEX1, LDH_D, NADH12, NADH6,PPS, THD2 and/or GLUDy 892 7 4.17527 0.08598 ADHEr, ATPS4r, FRD and/orSUCD4, HEX1, LDH_D, MALS, THD2 and/or GLUDy 893 7 4.17527 0.08598 ADHEr,ATPS4r, FRD and/or SUCD4, HEX1, ICL, LDH_D, THD2 and/or GLUDy 894 74.17215 0.10398 ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK, THD2 and/or GLUDy895 7 4.15042 0.11229 ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, THD2and/or GLUDy 896 7 4.14938 0.11268 ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK,THD2 and/or GLUDy 897 7 4.141 0.08314 ADHEr, ATPS4r, FRD and/or SUCD4,HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy 898 7 4.10258 0.11665 ACKr and/orPTAr, ADHEr, ATPS4r, LDH_D, PPCK, PYK, SUCOAS 899 7 4.08743 0.09886 ACKrand/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy 900 74.04907 0.15099 ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, PPCK 901 74.03008 0.1761 ADHEr, ASPT, GLCpts, LDH_D, MDH, PPCK, RPE 902 7 4.013980.06136 ADHEr, ATPS4r, LDH_D, PFLi, PGI, PPCK, THD2 and/or GLUDy 903 74.00698 0.18592 ADHEr, ASPT, LDH_D, MDH, NADH6, PPCK, RPE 904 7 4.001280.18834 ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, RPE 905 7 3.952050.10694 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGDH, PGI, SUCOAS 906 73.94203 0.1079 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGI, SUCOAS, TAL907 7 3.93273 0.1088 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGI, RPE,SUCOAS 908 7 3.90038 0.04761 ADHEr, GLCpts, LDH_D, NADH6, PGI, PPCK,THD2 and/or GLUDy 909 7 3.8947 0.05032 ADHEr, FUM, GLCpts, LDH_D, NADH6,PGI, THD2 and/or GLUDy 910 7 3.8947 0.05032 ADHEr, GLCpts, LDH_D, MDH,NADH6, PGI, THD2 and/or GLUDy 911 7 3.88125 0.03656 ACKr and/or PTAr,ADHEr, GLCpts, LDH_D, NADH6, PPCK, THD2 and/or GLUDy 912 7 3.866240.19846 ACKr and/or PTAr, ADHEr, CITL, LDH_D, NADH12, NADH6, THD2 and/orGLUDy 913 7 3.82356 0.31976 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PPS, THD2and/or GLUDy 914 7 3.81228 0.08972 ADHEr, EDA and/or PGDHY, LDH_D, MDH,NADH6, PYK, THD2 and/or GLUDy 915 7 3.81228 0.08972 ADHEr, LDH_D, MDH,NADH6, PGL and/or G6PDHy, PYK, THD2 and/or GLUDy 916 7 3.77115 0.10937ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK, THD2 and/or GLUDy 917 7 3.72910.12947 ADHEr, LDH_D, MDH, NADH12, NADH6, PYK, THD2 and/or GLUDy 918 73.7245 0.13167 ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, THD2 and/orGLUDy 919 7 3.68994 0.14818 ADHEr, GLCpts, LDH_D, NADH12, NADH6, PPCK,THD2 and/or GLUDy 920 7 3.67651 0.1546 ADHEr, LDH_D, MDH, NADH6, PGLand/or G6PDHy, PPCK, PYK 921 7 3.67651 0.1546 ADHEr, EDA and/or PGDHY,LDH_D, MDH, NADH6, PPCK, PYK 922 7 3.64497 0.19156 ADHEr, FUM, GLCpts,LDH_D, NADH6, RPE, THD2 and/or GLUDy 923 7 3.60739 0.21183 ADHEr, FUM,LDH_D, NADH12, NADH6, RPE, THD2 and/or GLUDy 924 7 3.41696 0.27865ADHEr, ALAR, FDH2, LDH_D, NADH12, NADH6, PRO1z 925 7 3.41696 0.27865ADHEr, DAAD, FDH2, LDH_D, NADH12, NADH6, PRO1z 926 7 3.40396 0.28486ADHEr, FUM, HEX1, LDH_D, NADH12, NADH6, THD2 and/or GLUDy 927 7 3.259090.04258 ADHEr, GLCpts, LDH_D, MDH, PGI, PPCK, THD2 and/or GLUDy 928 73.25909 0.04258 ADHEr, FUM, GLCpts, LDH_D, PGI, PPCK, THD2 and/or GLUDy929 7 3.2372 0.06431 ADHEr, FBP, LDH_D, MDH, PGI, PPCK, THD2 and/orGLUDy 930 7 3.2372 0.06431 ADHEr, FBP, FUM, LDH_D, PGI, PPCK, THD2and/or GLUDy 931 7 3.23399 0.06432 ADHEr, FUM, LDH_D, PGI, PPCK, RPE,THD2 and/or GLUDy 932 7 3.23399 0.06432 ADHEr, LDH_D, MDH, PGI, PPCK,RPE, THD2 and/or GLUDy 933 7 3.22783 0.06435 ADHEr, FUM, LDH_D, PGI,PPCK, TAL, THD2 and/or GLUDy 934 7 3.22783 0.06435 ADHEr, LDH_D, MDH,PGI, PPCK, TAL, THD2 and/or GLUDy 935 7 3.01859 0.19992 ADHEr, ASPT,FUM, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy 936 7 3.00174 0.19757 ADHEr,FRD and/or SUCD4, LDH_D, MALS, PPS, RPE, THD2 and/or GLUDy 937 7 3.001740.19757 ADHEr, FRD and/or SUCD4, ICL, LDH_D, PPS, RPE, THD2 and/or GLUDy938 7 2.96091 0.22716 ADHEr, FUM, GLUDy, HEX1, LDH_D, TAL, THD2 and/orGLUDy 939 7 2.93716 0.22721 ADHEr, FUM, GLU5K, GLUDy, HEX1, LDH_D, THD2and/or GLUDy 940 7 2.93716 0.22721 ADHEr, FUM, G5SD, GLUDy, HEX1, LDH_D,THD2 and/or GLUDy 941 7 2.93711 0.22724 ADHEr, ASNS2, FUM, GLUDy, HEX1,LDH_D, THD2 and/or GLUDy 942 7 2.7731 0.32129 ADHEr, ASNS2, FUM, GLU5K,HEX1, LDH_D, THD2 and/or GLUDy 943 7 2.7731 0.32129 ADHEr, ASNS2, FUM,G5SD, HEX1, LDH_D, THD2 and/or GLUDy 944 7 2.68652 0.29222 ADHEr,ATPS4r, GLUDy, HEX1, LDH_D, RPE, THD2 and/or GLUDy 945 7 2.62059 0.29297ADHEr, ATPS4r, GLUDy, HEX1, LDH_D, TAL, THD2 and/or GLUDy 946 7 2.359910.29396 ACKr and/or PTAr, ADHEr, FUM, LDH_D, RPE, SUCOAS, THD2 and/orGLUDy 947 7 2.35766 0.29368 ACKr and/or PTAr, ADHEr, FUM, LDH_D, SUCOAS,TAL, THD2 and/or GLUDy 948 7 2.3552 0.29337 ACKr and/or PTAr, ADHEr,FUM, LDH_D, PGL and/or G6PDHy, SUCOAS, THD2 and/or GLUDy 949 7 2.35520.29337 ACKr and/or PTAr, ADHEr, FUM, LDH_D, PGDH, SUCOAS, THD2 and/orGLUDy 950 7 1.91133 0.2766 ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, PGLand/or G6PDHy, SUCOAS 951 7 1.91133 0.2766 ACKr and/or PTAr, ADHEr, FUM,LDH_D, MDH, PGDH, SUCOAS 952 7 1.91133 0.2766 ACKr and/or PTAr, ADHEr,ICL, LDH_D, MDH, PGDH, SUCOAS 953 7 1.91133 0.2766 ACKr and/or PTAr,ADHEr, LDH_D, MALS, MDH, PGL and/or G6PDHy, SUCOAS 954 7 1.91133 0.2766ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, PGL and/or G6PDHy, SUCOAS 9557 1.91133 0.2766 ACKr and/or PTAr, ADHEr, LDH_D, MALS, MDH, PGDH, SUCOAS956 7 1.86676 0.2774 ACKr and/or PTAr, ADHEr, FUM, LDH_D, MDH, SUCOAS,TAL 957 7 1.86676 0.2774 ACKr and/or PTAr, ADHEr, LDH_D, MALS, MDH,SUCOAS, TAL 958 7 1.86676 0.2774 ACKr and/or PTAr, ADHEr, ICL, LDH_D,MDH, SUCOAS, TAL 959 7 1.8259 0.27813 ACKr and/or PTAr, ADHEr, LDH_D,MALS, MDH, RPE, SUCOAS 960 7 1.8259 0.27813 ACKr and/or PTAr, ADHEr,FUM, LDH_D, MDH, RPE, SUCOAS 961 7 1.8259 0.27813 ACKr and/or PTAr,ADHEr, ICL, LDH_D, MDH, RPE, SUCOAS 962 7 1.11652 0.25888 ADHEr, ASPT,FUM, LDH_D, MALS, PPS, THD2 and/or GLUDy 963 7 1.11652 0.25888 ADHEr,ASPT, FUM, ICL, LDH_D, PPS, THD2 and/or GLUDy 964 5 7.09275 0.11213ADHEr, ASPT, LDH_D, MDH, PFLi 965 5 6.00696 0.20464 ADHEr, ATPS4r,LDH_D, NADH6, PFLi 966 5 5.40138 0.11184 ADHEr, LDH_D, NADH6, PFLi, PGI967 5 2.59719 0.29318 ADHEr, FUM, HEX1, LDH_D, PFLi 968 5 2.574880.22587 ADHEr, HEX1, LDH_D, PFLi, PPCK 969 5 0.16844 0.19119 ADHEr,ASPT, FUM, LDH_D, PFLi 970 6 7.39516 0.07475 ADHEr, ASPT, GLCpts, LDH_D,MDH, PFLi 971 6 7.007 0.09285 ACKr and/or PTAr, ADHEr, LDH_D, NADH6,PFLi, PGI 972 6 6.90414 0.13454 ADHEr, ATPS4r, GLCpts, LDH_D, NADH6,PFLi 973 6 6.57261 0.1606 ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi 9746 6.00781 0.09114 ADHEr, LDH_D, MDH, PFLi, PGI, PPCK 975 6 6.007810.09114 ADHEr, FUM, LDH_D, PFLi, PGI, PPCK 976 6 5.39072 0.10901 ADHEr,LDH_D, NADH12, NADH6, PFLi, PPCK 977 6 5.38861 0.10972 ADHEr, FUM,LDH_D, NADH6, PFLi, PPCK 978 6 5.38861 0.10972 ADHEr, LDH_D, MDH, NADH6,PFLi, PPCK 979 6 5.06553 0.21856 ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi980 6 5.00212 0.2515 ADHEr, HEX1, LDH_D, NADH6, PFLi, RPE 981 6 4.983670.25221 ADHEr, HEX1, LDH_D, NADH6, PFLi, TAL 982 6 4.96609 0.25206ADHEr, GLU5K, HEX1, LDH_D, NADH6, PFLi 983 6 4.96609 0.25206 ADHEr,G5SD, HEX1, LDH_D, NADH6, PFLi 984 6 4.96599 0.2521 ADHEr, ASNS2, HEX1,LDH_D, NADH6, PFLi 985 6 4.95562 0.24186 ADHEr, EDA and/or PGDHY, LDH_D,MDH, PFLi, THD2 and/or GLUDy 986 6 2.71835 0.29123 ADHEr, FUM, HEX1,LDH_D, PFLi, RPE 987 6 2.66849 0.22473 ADHEr, HEX1, LDH_D, PFLi, PPCK,RPE 988 6 2.66076 0.29216 ADHEr, FUM, HEX1, LDH_D, PFLi, TAL 989 61.85699 0.28717 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PFLi, SUCOAS 9906 1.63724 0.03081 ADHEr, FUM, LDH_D, PFLi, PGI, THD2 and/or GLUDy 991 61.63724 0.03081 ADHEr, LDH_D, MDH, PFLi, PGI, THD2 and/or GLUDy 992 61.38263 0.33268 ADHEr, ATPS4r, LDH_D, NADH12, PFLi, THD2 and/or GLUDy993 6 0.25855 0.07515 ADHEr, ASPT, ATPS4r, FUM, LDH_D, PFLi 994 77.86642 0.01651 ADHEr, ASPT, LDH_D, MDH, NADH6, PFLi, PYK 995 7 7.715090.0515 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGI, PPS 996 7 7.70416 0.03656ADHEr, ASPT, ATPS4r, LDH_D, MDH, PFLi, PPCK 997 7 7.48515 0.04452 ADHEr,EDA and/or PGDHY, GLCpts, LDH_D, PFLi, PGI, PPCK 998 7 7.44181 0.09832ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi 999 7 7.066560.04513 ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi 1000 7 7.008110.04002 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PPCK 1001 7 7.008110.04002 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PPCK 1002 7 7.005320.07752 ADHEr, ATPS4r, FUM, LDH_D, NADH12, NADH6, PFLi 1003 7 6.991050.04159 ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, PPCK 1004 7 6.966950.09776 ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PFLi 1005 7 6.693450.1479 ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy 1006 76.49322 0.12467 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi, PPS 1007 76.43565 0.1244 ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi1008 7 6.21891 0.18801 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGDH, PPS 10097 6.21891 0.18801 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PGL and/or G6PDHy,PPS 1010 7 6.21459 0.18835 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPS, TAL1011 7 6.21064 0.18866 ADHEr, ATPS4r, LDH_D, NADH6, PFLi, PPS, RPE 10127 6.16665 0.08397 ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, PGI 1013 76.13458 0.01447 ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI, PPCK 1014 76.13051 0.01457 ADHEr, LDH_D, MDH, NADH6, PFLi, PGI, PPCK 1015 7 6.130510.01457 ADHEr, FUM, LDH_D, NADH6, PFLi, PGI, PPCK 1016 7 5.49961 0.07232ADHEr, GLCpts, LDH_D, NADH12, NADH6, PFLi, PPCK 1017 7 5.49821 0.07279ADHEr, FUM, GLCpts, LDH_D, NADH6, PFLi, PPCK 1018 7 5.49821 0.07279ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, PPCK 1019 7 5.22113 0.16614ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, THD2 and/or GLUDy 1020 75.17418 0.18196 ADHEr, FUM, HEX1, LDH_D, NADH12, NADH6, PFLi 1021 75.15097 0.18978 ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, THD2 and/orGLUDy 1022 7 4.01417 0.12149 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, THD2and/or GLUDy 1023 7 3.75439 0.27459 ADHEr, ATPS4r, FUM, HEX1, LDH_D,PFLi, PPS 1024 7 3.75439 0.27459 ADHEr, ATPS4r, HEX1, LDH_D, MDH, PFLi,PPS 1025 7 2.64048 0.21814 ADHEr, HEX1, LDH_D, PFLi, PGL and/or G6PDHy,PPCK, TAL 1026 7 2.64048 0.21814 ADHEr, HEX1, LDH_D, PFLi, PGDH, PPCK,TAL 1027 7 2.62716 0.22497 ADHEr, GLYCL, HEX1, LDH_D, PFLi, PPCK, TAL1028 7 1.5697 0.24425 ACKr and/or PTAr, ACS, ADHEr, LDH_D, MDH, PFLi,THD2 and/or GLUDy 1029 7 1.09053 0.33566 ADHEr, ATPS4r, FUM, LDH_D,PFLi, PGDH, PPS 1030 7 1.09053 0.33566 ADHEr, ATPS4r, FUM, LDH_D, PFLi,PGL and/or G6PDHy, PPS 1031 7 0.78572 0.33663 ADHEr, ATPS4r, FUM, LDH_D,PFLi, PPS, TAL 1032 7 0.50621 0.33751 ADHEr, ATPS4r, FUM, LDH_D, PFLi,PPS, RPE 1033 8 7.88716 0.0204 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D,MDH, NADH12, NADH6, PFLi 1034 8 7.88653 0.02051 ADHEr, ATPS4r, LDH_D,MDH, NADH12, NADH6, PFLi, POX 1035 8 7.88026 0.02165 ADHEr, GLCpts,LDH_D, MDH, NADH12, NADH6, PFLi, THD2 and/or GLUDy 1036 8 7.841040.02874 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, NADH6, PFLi, PGI, PPS1037 8 7.80925 0.03448 ADHEr, ATPS4r, FUM, LDH_D, NADH12, NADH6, PFLi,PGI 1038 8 7.80925 0.03448 ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6,PFLi, PGI 1039 8 7.67439 0.05735 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D,MDH, NADH12, NADH6, PFLi 1040 8 7.42051 0.09374 ADHEr, ATPS4r, FUM,GLCpts, LDH_D, NADH6, PFLi, PPS 1041 8 7.32674 0.0933 ACKr and/or PTAr,ADHEr, ATPS4r, FUM, GLCpts, LDH_D, NADH6, PFLi 1042 8 6.93593 0.04641ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, PPCK, THD2 and/or GLUDy 1043 86.58791 0.09796 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi, PGL and/orG6PDHy, PPS 1044 8 6.58791 0.09796 ADHEr, ATPS4r, HEX1, LDH_D, NADH6,PFLi, PGDH, PPS 1045 8 6.5802 0.09979 ADHEr, ATPS4r, HEX1, LDH_D, NADH6,PFLi, PPS, TAL 1046 8 6.57291 0.10152 ADHEr, ATPS4r, HEX1, LDH_D, NADH6,PFLi, PPS, RPE 1047 8 5.75656 0.15131 ADHEr, HEX1, LDH_D, NADH12, NADH6,PFLi, PPS, THD2 and/or GLUDy 1048 8 5.74602 0.16073 ADHEr, ATPS4r, HEX1,LDH_D, NADH12, PFLi, PPS, THD2 and/or GLUDy 1049 8 5.67321 0.16148 ACKrand/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, THD2 and/or GLUDy1050 8 5.60169 0.03793 ADHEr, FUM, LDH_D, NADH12, NADH6, PFLi, PPCK, PYK1051 8 5.60169 0.03793 ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi, PPCK, PYK1052 8 5.56692 0.04965 ADHEr, EDA and/or PGDHY, LDH_D, MDH, NADH6, PFLi,PPCK, PYK 1053 8 5.56692 0.04965 ADHEr, LDH_D, MDH, NADH6, PFLi, PGLand/or G6PDHy, PPCK, PYK 1054 8 5.50925 0.07241 ADHEr, ATPS4r, HEX1,LDH_D, PFLi, PPCK, RPE, THD2 and/or GLUDy 1055 8 5.50597 0.07191 ADHEr,ATPS4r, HEX1, LDH_D, PFLi, PPCK, TAL, THD2 and/or GLUDy 1056 8 5.502420.07138 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGL and/or G6PDHy, PPCK, THD2and/or GLUDy 1057 8 5.50242 0.07138 ADHEr, ATPS4r, HEX1, LDH_D, PFLi,PGDH, PPCK, THD2 and/or GLUDy 1058 8 5.44507 0.0907 ADHEr, HEX1, LDH_D,NADH12, NADH6, PFLi, PRO1z, THD2 and/or GLUDy 1059 8 5.37306 0.11496ADHEr, DAAD, LDH_D, MDH, NADH12, NADH6, PFLi, PRO1z 1060 8 5.373060.11496 ADHEr, ALAR, LDH_D, MDH, NADH12, NADH6, PFLi, PRO1z 1061 85.33606 0.12742 ADHEr, FUM, LDH_D, MDH, NADH12, NADH6, PFLi, PYK 1062 85.17764 0.18079 ADHEr, DAAD, HEX1, LDH_D, NADH12, NADH6, PFLi, PRO1z1063 8 5.17764 0.18079 ADHEr, ALAR, HEX1, LDH_D, NADH12, NADH6, PFLi,PRO1z 1064 8 5.17492 0.2398 ACKr and/or PTAr, ACS, ADHEr, EDA and/orPGDHY, LDH_D, MDH, PFLi, THD2 and/or GLUDy 1065 8 4.65995 0.20434 ADHEr,ATPS4r, HEX1, LDH_D, MDH, PFLi, PPS, RPE 1066 8 4.6123 0.20163 ADHEr,ATPS4r, HEX1, LDH_D, MDH, PFLi, PPS, TAL 1067 8 4.56149 0.19874 ADHEr,ATPS4r, HEX1, LDH_D, MDH, PFLi, PGDH, PPS 1068 8 4.13777 0.23628 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PFLi, PPS, RPE 1069 8 4.06785 0.23403 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PFLi, PPS, TAL 1070 8 4.04062 0.10178 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PDH, PFLi, PPS 1071 8 4.04062 0.10178 ADHEr,ATPS4r, HEX1, LDH_D, MDH, PDH, PFLi, PPS 1072 8 3.9927 0.23161 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PFLi, PGL and/or G6PDHy, PPS 1073 8 3.99270.23161 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi, PGDH, PPS 1074 8 2.036370.11796 ACKr and/or PTAr, ADHEr, FBP, LDH_D, MDH, PFLi, PGDH, THD2and/or GLUDy 1075 8 1.98516 0.11932 ACKr and/or PTAr, ADHEr, FBP, LDH_D,MDH, PFLi, TAL, THD2 and/or GLUDy 1076 8 1.93743 0.12058 ACKr and/orPTAr, ADHEr, FBP, LDH_D, MDH, PFLi, RPE, THD2 and/or GLUDy 1077 81.76983 0.24401 ACKr and/or PTAr, ACS, ADHEr, LDH_D, MDH, PFLi, PGI,THD2 and/or GLUDy 1078 8 1.5107 0.31956 ADHEr, ATPS4r, LDH_D, NADH12,PFLi, PGL and/or G6PDHy, TAL, THD2 and/or GLUDy 1079 8 1.5107 0.31956ADHEr, ATPS4r, LDH_D, NADH12, PFLi, PGDH, TAL, THD2 and/or GLUDy 1080 81.45644 0.3201 ADHEr, ATPS4r, FUM, LDH_D, PFLi, PGDH, PPS, TAL 1081 81.45644 0.3201 ADHEr, ATPS4r, FUM, LDH_D, PFLi, PGL and/or G6PDHy, PPS,TAL 1082 3 0.165681142 0.248770484 ADHEr, LDH_D, PFK and/or FBA and/orTPI 1083 3 2.792461067 0.310192487 ADHEr, FRD and/or SUCD4, LDH_D 1084 40.245091981 0.278197481 ADHEr, ASPT, FUM, LDH_D 1085 4 1.1636078130.384018912 ADHEr, ATPS4r, CBMK2, LDH_D 1086 4 2.27265597 0.332308612ADHEr, ATPS4r, LDH_D, NADH6 1087 4 1.260046787 0.384504476 ADHEr,ATPS4r, LDH_D, TKT1 1088 4 1.454891201 0.378757536 ADHEr, ATPS4r, LDH_D,TKT2 1089 4 2.042634808 0.153142196 ADHEr, HEX1, LDH_D, PFK and/or FBAand/or TPI 1090 4 2.041701304 0.149789093 ADHEr, LDH_D, PFK and/or FBAand/or TPI, PPCK 1091 4 0.270967943 0.244721556 ADHEr, LDH_D, PFK and/orFBA and/or TPI, TKT2 1092 4 2.907366868 0.244293538 ADHEr, FUM, LDH_D,PPCK 1093 4 0.363337262 0.340044232 ADHEr, HEX1, LDH_D, MDH 1094 42.907366868 0.244293538 ADHEr, LDH_D, MDH, PPCK 1095 4 2.0850824790.31290916 ADHEr, LDH_D, ME2, NADH6 1096 4 2.881545955 0.259101938ADHEr, FRD and/or SUCD4, LDH_D, ME2 1097 4 3.647199605 0.168610416ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI 1098 4 2.7745955690.320438424 ADHEr, FDH2, LDH_D, NADH6 1099 4 3.335904914 0.317384404ADHEr, FUM, LDH_D, NADH6 1100 4 3.331989713 0.319255557 ADHEr, HEX1,LDH_D, NADH6 1101 4 2.224731454 0.327457036 ADHEr, LDH_D, NADH6, TKT21102 4 0.23734264 0.246014656 ADHEr, LDH_D, PFK and/or FBA and/or TPI,RPE 1103 4 0.454138927 0.26931886 ADHEr, LDH_D, PPCK, TKT2 1104 40.413317212 0.38682004 ADHEr, HEX1, LDH_D, PPS 1105 4 2.8602946820.306273023 ADHEr, FRD and/or SUCD4, LDH_D, RPE 1106 4 2.8088407220.300798688 ADHEr, FRD and/or SUCD4, FUM, LDH_D 1107 4 2.8175748130.295789651 ADHEr, FRD and/or SUCD4, HEX1, LDH_D 1108 4 2.9161376960.239263432 ADHEr, FRD and/or SUCD4, LDH_D, MDH 1109 4 2.9738656520.206156212 ADHEr, FRD and/or SUCD4, LDH_D, PPCK 1110 4 2.8920502390.30443817 ADHEr, FRD and/or SUCD4, LDH_D, TKT2 1111 4 0.2033600990.247321495 ADHEr, LDH_D, PFK and/or FBA and/or TPI, TAL 1112 42.117903686 0.331070152 ADHEr, LDH_D, NADH6, TAL 1113 4 0.348849920.271004016 ADHEr, LDH_D, PPCK, TAL 1114 4 2.828154021 0.308130129ADHEr, FRD and/or SUCD4, LDH_D, TAL 1115 4 0.203360099 0.247321495ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT1 1116 4 2.1179036860.331070152 ADHEr, LDH_D, NADH6, TKT1 1117 4 0.34884992 0.271004016ADHEr, LDH_D, PPCK, TKT1 1118 4 2.828154021 0.308130129 ADHEr, FRDand/or SUCD4, LDH_D, TKT1 1119 5 3.948360602 0.222825522 ADHEr, ASPT,LDH_D, MDH, TKT2 1120 5 3.966967048 0.182343827 ADHEr, ASPT, FRD and/orSUCD4, LDH_D, MDH 1121 5 3.752898719 0.126622688 ADHEr, ATPS4r, FRDand/or SUCD4, LDH_D, ME2 1122 5 4.408716092 0.258053531 ADHEr, ATPS4r,LDH_D, MDH, THD2 and/or GLUDy 1123 5 6.415221004 0.039824926 ADHEr,FADH4, LDH_D, MDH, THD2 and/or GLUDy 1124 5 2.983060356 0.241650921ADHEr, FUM, LDH_D, PPCK, TKT2 1125 5 2.997718029 0.231538695 ADHEr, FUM,LDH_D, THD2 and/or GLUDy, TKT2 1126 5 2.937396425 0.227071464 ADHEr,FUM, HEX1, LDH_D, THD2 and/or GLUDy 1127 5 3.001461104 0.190330107ADHEr, FRD and/or SUCD4, FUM, LDH_D, THD2 and/or GLUDy 1128 52.935886887 0.22793719 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, THD2 and/orGLUDy 1129 5 3.081083028 0.144666606 ADHEr, FRD and/or SUCD4, LDH_D,PPCK, THD2 and/or GLUDy 1130 5 2.988449413 0.197792365 ADHEr, FRD and/orSUCD4, LDH_D, PRO1z, THD2 and/or GLUDy 1131 5 2.948350239 0.234817401ADHEr, FUM, LDH_D, TAL, THD2 and/or GLUDy 1132 5 3.101419157 0.133003752ADHEr, GLUDy, LDH_D, MDH, THD2 and/or GLUDy 1133 5 2.9483502390.234817401 ADHEr, FUM, LDH_D, THD2 and/or GLUDy, TKT1 1134 52.983060356 0.241650921 ADHEr, LDH_D, MDH, PPCK, TKT2 1135 5 2.9161376960.239263432 ADHEr, FRD and/or SUCD4, FUM, LDH_D, ME2 1136 5 3.7117873880.165696592 ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TKT2 1137 53.457829994 0.311699463 ADHEr, FUM, LDH_D, NADH6, TKT2 1138 53.451407023 0.315392099 ADHEr, HEX1, LDH_D, NADH6, TKT2 1139 53.543757983 0.262298527 ADHEr, LDH_D, MDH, NADH6, TKT2 1140 53.445461549 0.265025086 ADHEr, FUM, LDH_D, ME2, NADH6 1141 5 3.6095672520.224464061 ADHEr, LDH_D, NADH6, PPCK, TKT2 1142 5 3.6911744320.166626529 ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, RPE 1143 53.065706296 0.153485225 ADHEr, FRD and/or SUCD4, FUM, LDH_D, PFK and/orFBA and/or TPI 1144 5 3.071383178 0.15022951 ADHEr, FRD and/or SUCD4,LDH_D, MDH, PFK and/or FBA and/or TPI 1145 5 5.367175665 0.178044995ADHEr, CBMK2, HEX1, LDH_D, PGI 1146 5 5.888703714 0.106589745 ADHEr, EDAand/or PGDHY, FRD and/or SUCD4, LDH_D, PGI 1147 5 3.1109192150.148780301 ADHEr, FRD and/or SUCD4, LDH_D, MDH, PGI 1148 5 2.9587950950.242498072 ADHEr, FUM, LDH_D, PPCK, RPE 1149 5 2.958795095 0.242498072ADHEr, LDH_D, MDH, PPCK, RPE 1150 5 2.936051582 0.257223558 ADHEr, FRDand/or SUCD4, LDH_D, ME2, RPE 1151 5 3.418935049 0.313512999 ADHEr, FUM,LDH_D, NADH6, RPE 1152 5 3.413158878 0.316629525 ADHEr, HEX1, LDH_D,NADH6, RPE 1153 5 2.967063614 0.237144547 ADHEr, FRD and/or SUCD4,LDH_D, MDH, RPE 1154 5 3.018085814 0.204109778 ADHEr, FRD and/or SUCD4,LDH_D, PPCK, RPE 1155 5 3.009552774 0.185689501 ADHEr, FRD and/or SUCD4,FUM, LDH_D, PPCK 1156 5 3.009552774 0.185689501 ADHEr, FRD and/or SUCD4,LDH_D, MDH, PPCK 1157 5 2.991038247 0.23614703 ADHEr, FRD and/or SUCD4,LDH_D, MDH, TKT2 1158 5 2.961772661 0.256337155 ADHEr, FRD and/or SUCD4,LDH_D, ME2, TKT2 1159 5 3.106778446 0.151550201 ADHEr, FRD and/or SUCD4,FUM, LDH_D, PGI 1160 5 3.04583421 0.164881948 ADHEr, FRD and/or SUCD4,LDH_D, PPCK, PYK 1161 5 3.038870487 0.203147899 ADHEr, FRD and/or SUCD4,LDH_D, PPCK, TKT2 1162 5 3.886386663 0.225053566 ADHEr, ASPT, LDH_D,MDH, TAL 1163 5 3.670328799 0.167566962 ADHEr, LDH_D, NADH6, PFK and/orFBA and/or TPI, TAL 1164 5 3.480501908 0.264053132 ADHEr, LDH_D, MDH,NADH6, TAL 1165 5 3.554829627 0.226273464 ADHEr, LDH_D, NADH6, PPCK, TAL1166 5 2.942885577 0.238150528 ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL1167 5 2.99710338 0.20508081 ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TAL1168 5 3.964901837 0.267840742 ADHEr, HEX1, LDH_D, MDH, THD2 and/orGLUDy 1169 5 3.00074032 0.229453637 ADHEr, LDH_D, MDH, THD2 and/orGLUDy, TKT2 1170 5 4.557091208 0.201359081 ADHEr, FRD and/or SUCD4,LDH_D, ME2, THD2 and/or GLUDy 1171 5 2.935925119 0.227915263 ADHEr,LDH_D, MDH, PGDH, THD2 and/or GLUDy 1172 5 2.97971305 0.22895456 ADHEr,LDH_D, MDH, RPE, THD2 and/or GLUDy 1173 5 2.958777052 0.228457649 ADHEr,LDH_D, MDH, TAL, THD2 and/or GLUDy 1174 5 3.886386663 0.225053566 ADHEr,ASPT, LDH_D, MDH, TKT1 1175 5 2.934359102 0.243351183 ADHEr, FUM, LDH_D,PPCK, TKT1 1176 5 2.934359102 0.243351183 ADHEr, LDH_D, MDH, PPCK, TKT11177 5 3.670328799 0.167566962 ADHEr, LDH_D, NADH6, PFK and/or FBAand/or TPI, TKT1 1178 5 3.379584856 0.31534776 ADHEr, FUM, LDH_D, NADH6,TKT1 1179 5 3.374609422 0.317876699 ADHEr, HEX1, LDH_D, NADH6, TKT1 11805 3.480501908 0.264053132 ADHEr, LDH_D, MDH, NADH6, TKT1 1181 53.554829627 0.226273464 ADHEr, LDH_D, NADH6, PPCK, TKT1 1182 52.942885577 0.238150528 ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT1 1183 52.99710338 0.20508081 ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT1 1184 52.958777052 0.228457649 ADHEr, LDH_D, MDH, THD2 and/or GLUDy, TKT1

TABLE 15 Knockout strain designs for increased production of adipate,showing yields of adipate and biomass. New Num Adipate. Design ID KOYield Biomass Rxnlist 1 3 5.47908 0.34054 ADHEr, LDH_D, NADH6 2 34.26684 0.28031 ADHEr, LDH_D, PPCK 3 3 2.76166 0.38773 ADHEr, ATPS4r,LDH_D 4 3 1.54669 0.25966 ADHEr, LDH_D, PGI 5 3 1.33726 0.37808 ADHEr,FUM, LDH_D 6 3 0.83242 0.38953 ADHEr, HEX1, LDH_D 7 4 7.52636 0.16586ADHEr, HEX1, LDH_D, PGI 8 4 6.84269 0.27849 ADHEr, LDH_D, NADH6, PFLi 94 6.5702 0.20631 ADHEr, ASPT, LDH_D, MDH 10 4 6.16994 0.16614 ADHEr,LDH_D, NADH6, PGI 11 4 6.0882 0.30766 ADHEr, LDH_D, MDH, THD2 and/orGLUDy 12 4 5.87407 0.22728 ADHEr, LDH_D, NADH6, PPCK 13 4 5.684060.28176 ADHEr, LDH_D, MDH, NADH6 14 4 5.62555 0.33704 ADHEr, LDH_D,NADH6, RPE 15 4 5.60596 0.30416 ADHEr, LDH_D, NADH6, THD2 and/or GLUDy16 4 5.55606 0.3387 ADHEr, LDH_D, NADH6, TAL 17 4 5.54557 0.32148 ADHEr,FUM, LDH_D, NADH6 18 4 5.16028 0.24175 ADHEr, LDH_D, PFLi, PPCK 19 44.91511 0.27067 ADHEr, LDH_D, PPCK, THD2 and/or GLUDy 20 4 4.35680.24592 ADHEr, GLCpts, LDH_D, PPCK 21 4 4.35358 0.27902 ADHEr, LDH_D,PPCK, RPE 22 4 4.34095 0.25198 ADHEr, FUM, LDH_D, PPCK 23 4 4.340950.25198 ADHEr, LDH_D, MDH, PPCK 24 4 4.28493 0.2734 ADHEr, FUM, LDH_D,THD2 and/or GLUDy 25 4 4.0889 0.34834 ADHEr, FUM, HEX1, LDH_D 26 43.25969 0.38161 ADHEr, ATPS4r, LDH_D, RPE 27 4 3.18007 0.37054 ADHEr,HEX1, LDH_D, THD2 and/or GLUDy 28 4 3.11658 0.34524 ADHEr, ATPS4r,LDH_D, THD2 and/or GLUDy 29 4 3.02411 0.3845 ADHEr, ATPS4r, LDH_D, TAL30 4 1.1506 0.38695 ADHEr, HEX1, LDH_D, RPE 31 4 0.99936 0.38818 ADHEr,HEX1, LDH_D, TAL 32 5 7.63281 0.14897 ADHEr, HEX1, LDH_D, PGI, THD2and/or GLUDy 33 5 7.60368 0.23979 ADHEr, ATPS4r, LDH_D, MDH, NADH6 34 57.25714 0.17876 ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy 35 5 7.210610.18996 ADHEr, LDH_D, MDH, PFLi, PPCK 36 5 7.21061 0.18996 ADHEr, FUM,LDH_D, PFLi, PPCK 37 5 7.11676 0.21254 ADHEr, LDH_D, MDH, NADH6, PFLi 385 7.10568 0.21521 ADHEr, FUM, LDH_D, PFLi, THD2 and/or GLUDy 39 5 6.97060.24771 ADHEr, LDH_D, NADH6, PFLi, THD2 and/or GLUDy 40 5 6.906750.27577 ADHEr, LDH_D, NADH6, PFLi, RPE 41 5 6.83498 0.1681 ADHEr, ASPT,GLCpts, LDH_D, MDH 42 5 6.80758 0.29402 ADHEr, ATPS4r, LDH_D, MDH, TAL43 5 6.79015 0.29114 ADHEr, ATPS4r, LDH_D, MDH, PGDH 44 5 6.767430.17785 ADHEr, ASPT, LDH_D, MDH, THD2 and/or GLUDy 45 5 6.76201 0.23293ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy 46 5 6.66991 0.23493 ADHEr,LDH_D, PFLi, PPCK, THD2 and/or GLUDy 47 5 6.66754 0.2967 ADHEr, ATPS4r,LDH_D, MDH, RPE 48 5 6.57746 0.21333 ADHEr, LDH_D, NADH6, PPCK, THD2and/or GLUDy 49 5 6.34963 0.21785 ADHEr, ATPS4r, LDH_D, NADH6, PPCK 50 56.225 0.14772 ADHEr, LDH_D, NADH6, PGI, THD2 and/or GLUDy 51 5 6.189820.20058 ADHEr, GLUDy, LDH_D, MDH, THD2 and/or GLUDy 52 5 6.18254 0.21218ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy 53 5 6.18254 0.21218 ADHEr,FUM, LDH_D, PPCK, THD2 and/or GLUDy 54 5 5.99792 0.19177 ADHEr, GLCpts,LDH_D, NADH6, PPCK 55 5 5.97051 0.22537 ADHEr, LDH_D, NADH6, PPCK, RPE56 5 5.92744 0.32733 ADHEr, ATPS4r, HEX1, LDH_D, NADH6 57 5 5.924720.22627 ADHEr, LDH_D, NADH6, PPCK, TAL 58 5 5.81113 0.24533 ADHEr,GLCpts, LDH_D, MDH, NADH6 59 5 5.80132 0.28014 ADHEr, LDH_D, MDH, NADH6,RPE 60 5 5.74556 0.28091 ADHEr, LDH_D, MDH, NADH6, TAL 61 5 5.742350.20973 ADHEr, ATPS4r, LDH_D, MDH, THD2 and/or GLUDy 62 5 5.735750.30136 ADHEr, LDH_D, NADH6, RPE, THD2 and/or GLUDy 63 5 5.72421 0.22208ADHEr, ATPS4r, LDH_D, MDH, PPCK 64 5 5.72421 0.22208 ADHEr, ATPS4r, FUM,LDH_D, PPCK 65 5 5.68542 0.31766 ADHEr, FUM, LDH_D, NADH6, RPE 66 55.67414 0.30269 ADHEr, LDH_D, NADH6, TAL, THD2 and/or GLUDy 67 5 5.628710.29764 ADHEr, FUM, HEX1, LDH_D, NADH6 68 5 5.61913 0.31947 ADHEr, FUM,LDH_D, NADH6, TAL 69 5 5.56433 0.06187 ADHEr, ATPS4r, EDA and/or PGDHY,LDH_D, PGI 70 5 5.53618 0.32417 ADHEr, HEX1, LDH_D, NADH6, THD2 and/orGLUDy 71 5 5.36114 0.24084 ADHEr, LDH_D, PFLi, PPCK, RPE 72 5 5.357320.32317 ADHEr, ATPS4r, FUM, HEX1, LDH_D 73 5 5.29193 0.25262 ADHEr,ATPS4r, FUM, LDH_D, THD2 and/or GLUDy 74 5 5.26552 0.24127 ADHEr, LDH_D,PFLi, PPCK, TAL 75 5 5.23886 0.36027 ADHEr, HEX1, LDH_D, PFLi, PPS 76 55.2123 0.3202 ADHEr, ATPS4r, HEX1, LDH_D, PFLi 77 5 5.20628 0.31113ADHEr, FUM, HEX1, LDH_D, PFLi 78 5 5.20628 0.31113 ADHEr, HEX1, LDH_D,MDH, PFLi 79 5 5.00885 0.16853 ADHEr, ATPS4r, LDH_D, PFLi, PGI 80 54.73325 0.11896 ADHEr, LDH_D, PGI, PPCK, THD2 and/or GLUDy 81 5 4.718710.12544 ADHEr, LDH_D, MDH, PGI, THD2 and/or GLUDy 82 5 4.71871 0.12544ADHEr, FUM, LDH_D, PGI, THD2 and/or GLUDy 83 5 4.46543 0.20438 ADHEr,GLCpts, LDH_D, MDH, THD2 and/or GLUDy 84 5 4.45303 0.23609 ADHEr, LDH_D,MDH, RPE, THD2 and/or GLUDy 85 5 4.44799 0.21105 ADHEr, LDH_D, MDH, PYK,THD2 and/or GLUDy 86 5 4.43313 0.21674 ADHEr, GLCpts, LDH_D, MDH, PPCK87 5 4.43313 0.21674 ADHEr, FUM, GLCpts, LDH_D, PPCK 88 5 4.432890.24479 ADHEr, GLCpts, LDH_D, PPCK, RPE 89 5 4.42239 0.22084 ADHEr,GLCpts, LDH_D, PPCK, THD2 and/or GLUDy 90 5 4.4202 0.25027 ADHEr, FUM,LDH_D, PPCK, RPE 91 5 4.4202 0.25027 ADHEr, LDH_D, MDH, PPCK, RPE 92 54.41922 0.25069 ADHEr, LDH_D, PPCK, RPE, THD2 and/or GLUDy 93 5 4.413480.22425 ADHEr, LDH_D, MDH, PPCK, PYK 94 5 4.41348 0.22425 ADHEr, FUM,LDH_D, PPCK, PYK 95 5 4.3742 0.27012 ADHEr, FUM, LDH_D, RPE, THD2 and/orGLUDy 96 5 4.34566 0.25018 ADHEr, FUM, GLCpts, LDH_D, THD2 and/or GLUDy97 5 4.22695 0.31979 ADHEr, ATPS4r, HEX1, LDH_D, MDH 98 5 4.212970.36989 ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or GLUDy 99 5 4.197990.34618 ADHEr, FUM, HEX1, LDH_D, RPE 100 5 4.15986 0.32121 ADHEr, FUM,HEX1, LDH_D, THD2 and/or GLUDy 101 5 4.14613 0.34721 ADHEr, FUM, HEX1,LDH_D, TAL 102 5 4.14548 0.36885 ADHEr, HEX1, LDH_D, RPE, THD2 and/orGLUDy 103 5 4.09366 0.36854 ADHEr, HEX1, LDH_D, TAL, THD2 and/or GLUDy104 5 4.03694 0.36821 ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, THD2 and/orGLUDy 105 5 4.03694 0.36821 ADHEr, HEX1, LDH_D, PGDH, THD2 and/or GLUDy106 5 3.55579 0.34037 ADHEr, ATPS4r, LDH_D, RPE, THD2 and/or GLUDy 107 53.35244 0.11579 ADHEr, FRD and/or SUCD4, LDH_D, PPS, THD2 and/or GLUDy108 5 3.34785 0.34268 ADHEr, ATPS4r, LDH_D, TAL, THD2 and/or GLUDy 109 52.86477 0.08801 ADHEr, EDA and/or PGDHY, FUM, LDH_D, PGI 110 5 1.418160.36252 ADHEr, FUM, LDH_D, PGL and/or G6PDHy, TAL 111 5 1.41816 0.36252ADHEr, FUM, LDH_D, PGDH, TAL 112 6 8.40974 0.12863 ADHEr, GLUDy, LDH_D,MDH, PFLi, THD2 and/or GLUDy 113 6 8.3656 0.13555 ADHEr, LDH_D, MDH,PFLi, PPCK, THD2 and/or GLUDy 114 6 8.3656 0.13555 ADHEr, FUM, LDH_D,PFLi, PPCK, THD2 and/or GLUDy 115 6 8.30583 0.11621 ACKr and/or PTAr,ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy 116 6 8.24494 0.15445 ADHEr,LDH_D, MDH, NADH6, PFLi, THD2 and/or GLUDy 117 6 7.99084 0.19426 ADHEr,ATPS4r, LDH_D, MDH, PFLi, THD2 and/or GLUDy 118 6 7.9508 0.20053 ADHEr,HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy 119 6 7.92831 0.12763 ADHEr,ATPS4r, LDH_D, MDH, NADH6, THD2 and/or GLUDy 120 6 7.91305 0.13531ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK 121 6 7.91305 0.13531 ADHEr,ATPS4r, FUM, LDH_D, NADH6, PPCK 122 6 7.87012 0.02274 ACKr and/or PTAr,ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy 123 6 7.7587 0.21695 ADHEr,LDH_D, MDH, NADH12, NADH6, THD2 and/or GLUDy 124 6 7.75003 0.03607ADHEr, ASPT, LDH_D, MDH, PGI, THD2 and/or GLUDy 125 6 7.74149 0.21948ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, NADH6 126 6 7.7368 0.0672ADHEr, LDH_D, PFLi, PGI, PPCK, THD2 and/or GLUDy 127 6 7.72197 0.07098ADHEr, LDH_D, MDH, PFLi, PGI, THD2 and/or GLUDy 128 6 7.72197 0.07098ADHEr, FUM, LDH_D, PFLi, PGI, THD2 and/or GLUDy 129 6 7.67273 0.08355ADHEr, LDH_D, NADH6, PFLi, PGI, THD2 and/or GLUDy 130 6 7.64843 0.19881ADHEr, GLCpts, LDH_D, MDH, NADH6, THD2 and/or GLUDy 131 6 7.618240.14232 ADHEr, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy 132 6 7.608250.2542 ADHEr, FUM, HEX1, LDH_D, PFLi, THD2 and/or GLUDy 133 6 7.54670.16899 ADHEr, ATPS4r, LDH_D, PFLi, PPCK, THD2 and/or GLUDy 134 67.48349 0.12429 ADHEr, FUM, LDH_D, NADH6, PFLi, THD2 and/or GLUDy 135 67.4685 0.07894 ADHEr, ASPT, LDH_D, MDH, PGL and/or G6PDHy, PYK 136 67.4685 0.07894 ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PYK 137 67.44298 0.08037 ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, THD2 and/orGLUDy 138 6 7.40415 0.13677 ADHEr, LDH_D, NADH6, PFLi, PPCK, PYK 139 67.4 0.14438 ADHEr, GLCpts, LDH_D, MDH, PFLi, THD2 and/or GLUDy 140 67.36241 0.15343 ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK 141 6 7.362410.15343 ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK 142 6 7.27527 0.1744ADHEr, ATPS4r, FUM, LDH_D, NADH6, THD2 and/or GLUDy 143 6 7.274480.17459 ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi 144 6 7.24864 0.17893ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, THD2 and/or GLUDy 145 67.24278 0.26146 ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy 146 67.20895 0.28496 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, THD2 and/or GLUDy 1476 7.18819 0.12101 ADHEr, ASPT, LDH_D, MDH, PYK, THD2 and/or GLUDy 148 67.17088 0.20914 ADHEr, LDH_D, MDH, PFLi, RPE, THD2 and/or GLUDy 149 67.15564 0.2082 ADHEr, LDH_D, MDH, PFLi, TAL, THD2 and/or GLUDy 150 67.1458 0.12325 ADHEr, ASPT, LDH_D, MDH, PPCK, THD2 and/or GLUDy 151 67.13909 0.20717 ADHEr, LDH_D, MDH, PFLi, PGDH, THD2 and/or GLUDy 152 67.12504 0.22071 ADHEr, LDH_D, PFLi, PPCK, RPE, THD2 and/or GLUDy 153 67.10894 0.21971 ADHEr, LDH_D, PFLi, PPCK, TAL, THD2 and/or GLUDy 154 67.09146 0.21863 ADHEr, LDH_D, PFLi, PGL and/or G6PDHy, PPCK, THD2 and/orGLUDy 155 6 7.09146 0.21863 ADHEr, LDH_D, PFLi, PGDH, PPCK, THD2 and/orGLUDy 156 6 7.0863 0.31638 ADHEr, HEX1, LDH_D, PFLi, PPS, THD2 and/orGLUDy 157 6 7.06893 0.22405 ADHEr, LDH_D, NADH12, NADH6, PFLi, THD2and/or GLUDy 158 6 7.054 0.1365 ADHEr, ASPT, LDH_D, MDH, NADH6, THD2and/or GLUDy 159 6 7.02653 0.24555 ADHEr, LDH_D, NADH6, PFLi, RPE, THD2and/or GLUDy 160 6 6.99568 0.14491 ADHEr, ASPT, GLCpts, LDH_D, MDH, THD2and/or GLUDy 161 6 6.92353 0.16046 ADHEr, ASPT, GLUDy, LDH_D, MDH, THD2and/or GLUDy 162 6 6.90243 0.27063 ADHEr, HEX1, LDH_D, NADH6, PFLi, TAL163 6 6.8314 0.18819 ADHEr, ATPS4r, LDH_D, MDH, PGL and/or G6PDHy, PPCK164 6 6.79251 0.30763 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PFLi 165 66.70257 0.21085 ADHEr, ATPS4r, LDH_D, NADH6, PGL and/or G6PDHy, PPCK 1666 6.70257 0.21085 ADHEr, ATPS4r, LDH_D, NADH6, PGDH, PPCK 167 6 6.667430.21154 ADHEr, ATPS4r, LDH_D, NADH6, PPCK, TAL 168 6 6.63518 0.21218ADHEr, ATPS4r, LDH_D, NADH6, PPCK, RPE 169 6 6.48093 0.19172 ADHEr,ATPS4r, FUM, LDH_D, PGL and/or G6PDHy, PPCK 170 6 6.48093 0.19172 ADHEr,ATPS4r, FUM, LDH_D, PGDH, PPCK 171 6 6.44865 0.19659 ADHEr, ATPS4r,GLCpts, LDH_D, PFLi, PPCK 172 6 6.44275 0.19249 ADHEr, ATPS4r, FUM,LDH_D, PPCK, TAL 173 6 6.40767 0.1932 ADHEr, ATPS4r, FUM, LDH_D, PPCK,RPE 174 6 6.33204 0.06396 ACKr and/or PTAr, ADHEr, LDH_D, NADH6, PPCK,THD2 and/or GLUDy 175 6 6.28774 0.02127 ADHEr, ATPS4r, EDA and/or PGDHY,LDH_D, PGI, THD2 and/or GLUDy 176 6 6.27764 0.32432 ADHEr, ATPS4r, HEX1,LDH_D, NADH6, PGL and/or G6PDHy 177 6 6.27764 0.32432 ADHEr, ATPS4r,HEX1, LDH_D, NADH6, PGDH 178 6 6.21873 0.32483 ADHEr, ATPS4r, HEX1,LDH_D, NADH6, TAL 179 6 6.16484 0.32529 ADHEr, ATPS4r, HEX1, LDH_D,NADH6, RPE 180 6 6.12301 0.15589 ADHEr, LDH_D, MDH, NADH6, PYK, THD2and/or GLUDy 181 6 6.09151 0.3086 ADHEr, FUM, HEX1, LDH_D, PPS, THD2and/or GLUDy 182 6 6.08785 0.16598 ADHEr, FUM, LDH_D, NADH6, PPCK, PYK183 6 6.08785 0.16598 ADHEr, LDH_D, MDH, NADH6, PPCK, PYK 184 6 6.079290.19015 ADHEr, GLCpts, LDH_D, NADH6, PPCK, RPE 185 6 6.0681 0.17164ADHEr, GLCpts, LDH_D, NADH6, PPCK, THD2 and/or GLUDy 186 6 6.043020.20189 ADHEr, LDH_D, NADH6, PPCK, RPE, THD2 and/or GLUDy 187 6 6.019110.18569 ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK 188 6 6.01911 0.18569ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK 189 6 5.98595 0.1952 ADHEr, FUM,GLCpts, LDH_D, NADH6, THD2 and/or GLUDy 190 6 5.93003 0.21123 ADHEr,FUM, LDH_D, NADH12, NADH6, THD2 and/or GLUDy 191 6 5.91322 0.24391ADHEr, GLCpts, LDH_D, MDH, NADH6, RPE 192 6 5.89537 0.30664 ADHEr,ATPS4r, HEX1, LDH_D, MDH, PFLi 193 6 5.86467 0.24459 ADHEr, GLCpts,LDH_D, MDH, NADH6, TAL 194 6 5.8616 0.12855 ADHEr, ATPS4r, LDH_D, MDH,PPCK, THD2 and/or GLUDy 195 6 5.8616 0.12855 ADHEr, ATPS4r, FUM, LDH_D,PPCK, THD2 and/or GLUDy 196 6 5.75652 0.29464 ADHEr, FUM, HEX1, LDH_D,NADH6, RPE 197 6 5.71417 0.25845 ADHEr, ATPS4r, LDH_D, MDH, PGL and/orG6PDHy, PPS 198 6 5.69588 0.29606 ADHEr, FUM, HEX1, LDH_D, NADH6, TAL199 6 5.53662 0.35787 ADHEr, HEX1, LDH_D, PFLi, PPS, RPE 200 6 5.47240.31504 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, RPE 201 6 5.46402 0.30945ADHEr, FUM, HEX1, LDH_D, PFLi, RPE 202 6 5.46402 0.30945 ADHEr, HEX1,LDH_D, MDH, PFLi, RPE 203 6 5.39509 0.35901 ADHEr, HEX1, LDH_D, PFLi,PPS, TAL 204 6 5.34939 0.31748 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, TAL 2056 5.34143 0.31025 ADHEr, HEX1, LDH_D, MDH, PFLi, TAL 206 6 5.341430.31025 ADHEr, FUM, HEX1, LDH_D, PFLi, TAL 207 6 5.07203 0.20521 ADHEr,LDH_D, MDH, PFLi, PGDH, PGI 208 6 5.07203 0.20521 ADHEr, FUM, LDH_D,PFLi, PGDH, PGI 209 6 5.04668 0.20656 ADHEr, FUM, LDH_D, PFLi, PGI, TAL210 6 5.04668 0.20656 ADHEr, LDH_D, MDH, PFLi, PGI, TAL 211 6 5.023270.2078 ADHEr, LDH_D, MDH, PFLi, PGI, RPE 212 6 5.02327 0.2078 ADHEr,FUM, LDH_D, PFLi, PGI, RPE 213 6 4.96889 0.09918 ACKr and/or PTAr,ADHEr, ATPS4r, LDH_D, PPCK, THD2 and/or GLUDy 214 6 4.92469 0.33322ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/or GLUDy 215 64.92469 0.33322 ADHEr, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy 216 64.8531 0.05616 ADHEr, LDH_D, MDH, PGI, PPCK, THD2 and/or GLUDy 217 64.8531 0.05616 ADHEr, FUM, LDH_D, PGI, PPCK, THD2 and/or GLUDy 218 64.80931 0.34014 ADHEr, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy 219 64.69982 0.3467 ADHEr, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy 220 64.69121 0.11806 ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, THD2 and/orGLUDy 221 6 4.64152 0.13706 ADHEr, FUM, GLCpts, LDH_D, PPCK, THD2 and/orGLUDy 222 6 4.64152 0.13706 ADHEr, GLCpts, LDH_D, MDH, PPCK, THD2 and/orGLUDy 223 6 4.6299 0.1415 ADHEr, LDH_D, MDH, PPCK, PYK, THD2 and/orGLUDy 224 6 4.6299 0.1415 ADHEr, FUM, LDH_D, PPCK, PYK, THD2 and/orGLUDy 225 6 4.60999 0.16834 ADHEr, LDH_D, MDH, PPCK, RPE, THD2 and/orGLUDy 226 6 4.60999 0.16834 ADHEr, FUM, LDH_D, PPCK, RPE, THD2 and/orGLUDy 227 6 4.52953 0.20307 ADHEr, GLCpts, LDH_D, MDH, RPE, THD2 and/orGLUDy 228 6 4.5125 0.21043 ADHEr, LDH_D, MDH, PYK, RPE, THD2 and/orGLUDy 229 6 4.50129 0.21526 ADHEr, GLCpts, LDH_D, MDH, PPCK, RPE 230 64.50129 0.21526 ADHEr, FUM, GLCpts, LDH_D, PPCK, RPE 231 6 4.490480.21993 ADHEr, GLCpts, LDH_D, PPCK, RPE, THD2 and/or GLUDy 232 6 4.482110.22354 ADHEr, FUM, LDH_D, PPCK, PYK, RPE 233 6 4.48211 0.22354 ADHEr,LDH_D, MDH, PPCK, PYK, RPE 234 6 4.42882 0.24654 ADHEr, FUM, GLCpts,LDH_D, RPE, THD2 and/or GLUDy 235 6 4.36373 0.24327 ADHEr, FUM, GLUDy,HEX1, LDH_D, THD2 and/or GLUDy 236 6 4.35757 0.26123 ADHEr, FUM, HEX1,LDH_D, TAL, THD2 and/or GLUDy 237 6 4.31718 0.27765 ADHEr, ASNS2, GLU5K,LDH_D, PPCK, TAL 238 6 4.31718 0.27765 ADHEr, ASNS2, G5SD, LDH_D, PPCK,TAL 239 6 4.2573 0.32058 ADHEr, FUM, HEX1, LDH_D, RPE, THD2 and/or GLUDy240 6 4.22017 0.33524 ADHEr, GLUDy, HEX1, LDH_D, RPE, THD2 and/or GLUDy241 6 4.17617 0.33499 ADHEr, GLUDy, HEX1, LDH_D, TAL, THD2 and/or GLUDy242 6 4.12454 0.33472 ADHEr, GLUDy, HEX1, LDH_D, PGDH, THD2 and/or GLUDy243 6 4.12454 0.33472 ADHEr, GLUDy, HEX1, LDH_D, PGL and/or G6PDHy, THD2and/or GLUDy 244 6 3.51205 0.21436 ADHEr, LDH_D, PFLi, PGI, PPS, THD2and/or GLUDy 245 6 3.23372 0.26787 ACKr and/or PTAr, ADHEr, ATPS4r, FUM,LDH_D, THD2 and/or GLUDy 246 6 2.95584 0.24016 ADHEr, FUM, LDH_D, PFLi,PGI, PPS 247 6 2.95584 0.24016 ADHEr, LDH_D, MDH, PFLi, PGI, PPS 248 79.10608 0.01953 ADHEr, GLUDy, LDH_D, MDH, PFLi, PPCK, THD2 and/or GLUDy249 7 9.10608 0.01953 ADHEr, FUM, GLUDy, LDH_D, PFLi, PPCK, THD2 and/orGLUDy 250 7 9.00855 0.03482 ADHEr, GLUDy, LDH_D, MDH, NADH6, PFLi, THD2and/or GLUDy 251 7 8.9957 0.03683 ADHEr, FUM, LDH_D, NADH6, PFLi, PPCK,THD2 and/or GLUDy 252 7 8.9957 0.03683 ADHEr, LDH_D, MDH, NADH6, PFLi,PPCK, THD2 and/or GLUDy 253 7 8.98751 0.03811 ADHEr, ATPS4r, HEX1,LDH_D, PFLi, PGI, THD2 and/or GLUDy 254 7 8.98549 0.03843 ADHEr, LDH_D,MDH, NADH12, NADH6, PFLi, THD2 and/or GLUDy 255 7 8.78817 0.06934 ADHEr,ATPS4r, GLUDy, LDH_D, MDH, PFLi, THD2 and/or GLUDy 256 7 8.78695 0.06953ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH, THD2 and/or GLUDy 257 7 8.783710.07004 ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL, THD2 and/or GLUDy 258 78.78071 0.07051 ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE, THD2 and/or GLUDy259 7 8.7615 0.07352 ADHEr, ATPS4r, LDH_D, MDH, PFLi, PPCK, THD2 and/orGLUDy 260 7 8.7615 0.07352 ADHEr, ATPS4r, FUM, LDH_D, PFLi, PPCK, THD2and/or GLUDy 261 7 8.5647 0.10435 ADHEr, GLUDy, HEX1, LDH_D, MDH, PFLi,THD2 and/or GLUDy 262 7 8.54703 0.10712 ADHEr, GLCpts, GLUDy, LDH_D,MDH, PFLi, THD2 and/or GLUDy 263 7 8.53424 0.10913 ADHEr, HEX1, LDH_D,MDH, PFLi, PPCK, THD2 and/or GLUDy 264 7 8.53424 0.10913 ADHEr, FUM,HEX1, LDH_D, PFLi, PPCK, THD2 and/or GLUDy 265 7 8.50919 0.10634 ACKrand/or PTAr, ADHEr, LDH_D, MDH, NADH12, NADH6, THD2 and/or GLUDy 266 78.50798 0.11324 ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK, THD2 and/or GLUDy267 7 8.50798 0.11324 ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK, THD2 and/orGLUDy 268 7 8.43629 0.12447 ADHEr, HEX1, LDH_D, MDH, NADH6, PFLi, THD2and/or GLUDy 269 7 8.38405 0.13266 ADHEr, GLCpts, LDH_D, MDH, NADH6,PFLi, THD2 and/or GLUDy 270 7 8.37888 0.03978 ADHEr, LDH_D, NADH12,NADH6, PFLi, PPCK, THD2 and/or GLUDy 271 7 8.29239 0.14702 ADHEr,ATPS4r, HEX1, LDH_D, MDH, PFLi, THD2 and/or GLUDy 272 7 8.27109 0.10802ADHEr, ATPS4r, GLUDy, LDH_D, MDH, NADH6, THD2 and/or GLUDy 273 7 8.252380.15328 ADHEr, ATPS4r, LDH_D, MDH, NADH12, PFLi, THD2 and/or GLUDy 274 78.24277 0.01559 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, NADH6, PGI, THD2and/or GLUDy 275 7 8.13676 0.03579 ADHEr, ATPS4r, GLCpts, LDH_D, MDH,NADH6, THD2 and/or GLUDy 276 7 8.112 0.11376 ADHEr, ATPS4r, FUM, LDH_D,NADH6, PPCK, THD2 and/or GLUDy 277 7 8.112 0.11376 ADHEr, ATPS4r, LDH_D,MDH, NADH6, PPCK, THD2 and/or GLUDy 278 7 8.02815 0.13154 ADHEr, ATPS4r,GLCpts, LDH_D, MDH, PFLi, THD2 and/or GLUDy 279 7 8.00984 0.13929 ADHEr,ATPS4r, LDH_D, NADH12, PFLi, PPCK, THD2 and/or GLUDy 280 7 8.00640.13951 ADHEr, ATPS4r, FUM, GLCpts, LDH_D, PFLi, PPCK 281 7 8.00640.13951 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, PFLi, PPCK 282 7 7.980070.18432 ADHEr, HEX1, LDH_D, MDH, NADH12, NADH6, THD2 and/or GLUDy 283 77.97463 0.15783 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, NADH6, THD2and/or GLUDy 284 7 7.90344 0.19562 ADHEr, GLCpts, LDH_D, MDH, NADH12,NADH6, THD2 and/or GLUDy 285 7 7.89808 0.01471 ADHEr, ASPT, GLCpts,LDH_D, MDH, PGI, THD2 and/or GLUDy 286 7 7.89024 0.02802 ADHEr, ATPS4r,LDH_D, PFLi, PGI, PPCK, THD2 and/or GLUDy 287 7 7.88378 0.02967 ADHEr,ATPS4r, LDH_D, MDH, PFLi, PGI, THD2 and/or GLUDy 288 7 7.88242 0.11605ADHEr, GLUDy, LDH_D, MDH, NADH12, NADH6, THD2 and/or GLUDy 289 7 7.876340.01837 ADHEr, ASPT, LDH_D, MDH, NADH6, PGL and/or G6PDHy, PYK 290 77.87634 0.01837 ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, NADH6, PYK291 7 7.84906 0.21647 ADHEr, ATPS4r, HEX1, LDH_D, NADH12, PFLi, THD2and/or GLUDy 292 7 7.84582 0.02225 ACKr and/or PTAr, ADHEr, ASPT, LDH_D,MDH, PPCK, THD2 and/or GLUDy 293 7 7.81872 0.2081 ADHEr, ATPS4r, EDAand/or PGDHY, LDH_D, MDH, NADH6, PPS 294 7 7.7627 0.0571 ADHEr, LDH_D,MDH, PFLi, PPCK, PYK, THD2 and/or GLUDy 295 7 7.7627 0.0571 ADHEr, FUM,LDH_D, PFLi, PPCK, PYK, THD2 and/or GLUDy 296 7 7.76232 0.02662 ADHEr,ATPS4r, LDH_D, NADH6, PGI, PPCK, THD2 and/or GLUDy 297 7 7.74907 0.0281ADHEr, ATPS4r, FUM, LDH_D, NADH6, PGI, THD2 and/or GLUDy 298 7 7.744440.06699 ACKr and/or PTAr, ADHEr, ATPS4r, GLCpts, LDH_D, MDH, NADH6 299 77.71368 0.10674 ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK, THD2 and/orGLUDy 300 7 7.69617 0.04384 ACKr and/or PTAr, ADHEr, ASPT, GLCpts,LDH_D, MDH, THD2 and/or GLUDy 301 7 7.69209 0.0741 ADHEr, LDH_D, MDH,NADH6, PFLi, PYK, THD2 and/or GLUDy 302 7 7.61698 0.05527 ADHEr, ASPT,LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy 303 7 7.58851 0.09902 ADHEr,FUM, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy 304 7 7.55893 0.07788ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, RPE, THD2 and/or GLUDy 305 77.55773 0.07778 ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, TAL, THD2and/or GLUDy 306 7 7.55641 0.07767 ACKr and/or PTAr, ADHEr, GLUDy,LDH_D, MDH, PGDH, THD2 and/or GLUDy 307 7 7.55604 0.07774 ADHEr, GLUDy,LDH_D, MDH, NADH6, PPCK, THD2 and/or GLUDy 308 7 7.52827 0.18666 ADHEr,ATPS4r, FUM, HEX1, LDH_D, NADH6, PPS 309 7 7.5043 0.12303 ACKr and/orPTAr, ADHEr, HEX1, LDH_D, MDH, PGDH, THD2 and/or GLUDy 310 7 7.503210.07168 ACKr and/or PTAr, ADHEr, ASPT, GLUDy, LDH_D, MDH, THD2 and/orGLUDy 311 7 7.50183 0.1239 ACKr and/or PTAr, ADHEr, HEX1, LDH_D, MDH,TAL, THD2 and/or GLUDy 312 7 7.49954 0.12472 ACKr and/or PTAr, ADHEr,HEX1, LDH_D, MDH, RPE, THD2 and/or GLUDy 313 7 7.44132 0.08061 ADHEr,ASPT, LDH_D, MDH, NADH6, PPCK, THD2 and/or GLUDy 314 7 7.43036 0.11712ADHEr, EDA and/or PGDHY, GLUDy, LDH_D, MDH, PPCK, THD2 and/or GLUDy 3157 7.38132 0.14888 ADHEr, LDH_D, NADH12, NADH6, PFLi, PRO1z, THD2 and/orGLUDy 316 7 7.37291 0.17014 ACKr and/or PTAr, ADHEr, ATPS4r, FUM, HEX1,LDH_D, THD2 and/or GLUDy 317 7 7.37059 0.09082 ADHEr, ASPT, GLCpts,LDH_D, MDH, PPCK, THD2 and/or GLUDy 318 7 7.3166 0.16445 ADHEr, HEX1,LDH_D, NADH12, NADH6, PFLi, THD2 and/or GLUDy 319 7 7.2905 0.10237ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6, THD2 and/or GLUDy 320 7 7.248640.17893 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, PGDH, THD2 and/orGLUDy 321 7 7.2288 0.11128 ADHEr, ASPT, GLUDy, LDH_D, MDH, PPCK, THD2and/or GLUDy 322 7 7.22725 0.1115 ADHEr, ASPT, LDH_D, MDH, NADH12,NADH6, THD2 and/or GLUDy 323 7 7.21589 0.1796 ADHEr, ATPS4r, EDA and/orPGDHY, LDH_D, MDH, TAL, THD2 and/or GLUDy 324 7 7.21263 0.18991 ADHEr,ATPS4r, EDA and/or PGDHY, LDH_D, MDH, PPCK, TAL 325 7 7.20753 0.1999ADHEr, GLUDy, LDH_D, PFLi, PPCK, RPE, THD2 and/or GLUDy 326 7 7.205070.13919 ADHEr, FUM, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy 327 77.20507 0.13919 ADHEr, FUM, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2and/or GLUDy 328 7 7.20053 0.0557 ADHEr, ATPS4r, FUM, GLCpts, LDH_D,PPCK, THD2 and/or GLUDy 329 7 7.20053 0.0557 ADHEr, ATPS4r, GLCpts,LDH_D, MDH, PPCK, THD2 and/or GLUDy 330 7 7.19596 0.14141 ADHEr, FUM,HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy 331 7 7.19261 0.19908 ADHEr,GLUDy, LDH_D, PFLi, PPCK, TAL, THD2 and/or GLUDy 332 7 7.18739 0.14349ADHEr, FUM, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy 333 7 7.185820.18022 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, RPE, THD2 and/orGLUDy 334 7 7.18081 0.1906 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH,PPCK, RPE 335 7 7.17639 0.19819 ADHEr, GLUDy, LDH_D, PFLi, PGDH, PPCK,THD2 and/or GLUDy 336 7 7.17639 0.19819 ADHEr, GLUDy, LDH_D, PFLi, PGLand/or G6PDHy, PPCK, THD2 and/or GLUDy 337 7 7.15844 0.21228 ADHEr, FUM,GLU5K, LDH_D, PFLi, RPE, THD2 and/or GLUDy 338 7 7.15844 0.21228 ADHEr,FUM, G5SD, LDH_D, PFLi, RPE, THD2 and/or GLUDy 339 7 7.15832 0.21231ADHEr, ASNS2, FUM, LDH_D, PFLi, RPE, THD2 and/or GLUDy 340 7 7.150230.12261 ADHEr, ASPT, GLUDy, LDH_D, MDH, NADH6, THD2 and/or GLUDy 341 77.14754 0.30973 ADHEr, GLUDy, HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy342 7 7.12499 0.05298 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PPCK,THD2 and/or GLUDy 343 7 7.12287 0.13074 ADHEr, ASPT, GLCpts, GLUDy,LDH_D, MDH, THD2 and/or GLUDy 344 7 7.12019 0.22193 ADHEr, LDH_D,NADH12, NADH6, PFLi, RPE, THD2 and/or GLUDy 345 7 7.11351 0.16642 ACKrand/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, MDH, THD2 and/or GLUDy 346 76.91657 0.2887 ADHEr, ATPS4r, HEX1, LDH_D, MDH, PGDH, PPS 347 7 6.899390.23701 ADHEr, ATPS4r, FUM, LDH_D, MDH, PGL and/or G6PDHy, PPS 348 76.89939 0.23701 ADHEr, ATPS4r, LDH_D, MALS, MDH, PGL and/or G6PDHy, PPS349 7 6.89939 0.23701 ADHEr, ATPS4r, ICL, LDH_D, MDH, PGL and/or G6PDHy,PPS 350 7 6.84379 0.18963 ADHEr, ATPS4r, EDA and/or PGDHY, GLUDy, LDH_D,MDH, THD2 and/or GLUDy 351 7 6.75658 0.30629 ADHEr, ATPS4r, FUM, HEX1,LDH_D, PPS, RPE 352 7 6.62545 0.08375 ACKr and/or PTAr, ADHEr, ATPS4r,GLUDy, LDH_D, MDH, THD2 and/or GLUDy 353 7 6.61503 0.30219 ADHEr,ATPS4r, FUM, HEX1, LDH_D, PPS, TAL 354 7 6.5251 0.02706 ACKr and/orPTAr, ADHEr, GLCpts, LDH_D, NADH6, PPCK, THD2 and/or GLUDy 355 7 6.500640.04761 ADHEr, GLCpts, LDH_D, NADH6, PGI, PPCK, THD2 and/or GLUDy 356 76.49117 0.05032 ADHEr, FUM, GLCpts, LDH_D, NADH6, PGI, THD2 and/or GLUDy357 7 6.49117 0.05032 ADHEr, GLCpts, LDH_D, MDH, NADH6, PGI, THD2 and/orGLUDy 358 7 6.46411 0.29781 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGL and/orG6PDHy, PPS 359 7 6.46411 0.29781 ADHEr, ATPS4r, FUM, HEX1, LDH_D, PGDH,PPS 360 7 6.37646 0.08322 ADHEr, FUM, LDH_D, NADH12, NADH6, PPCK, THD2and/or GLUDy 361 7 6.37646 0.08322 ADHEr, LDH_D, MDH, NADH12, NADH6,PPCK, THD2 and/or GLUDy 362 7 6.33505 0.15513 ADHEr, ATPS4r, FUM, LDH_D,NADH6, PGDH, PGI 363 7 6.30836 0.31643 ADHEr, HEX1, LDH_D, MALS, PGLand/or G6PDHy, PPS, THD2 and/or GLUDy 364 7 6.30836 0.31643 ADHEr, HEX1,ICL, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/or GLUDy 365 7 6.308360.31643 ADHEr, HEX1, LDH_D, MALS, PGDH, PPS, THD2 and/or GLUDy 366 76.30836 0.31643 ADHEr, HEX1, ICL, LDH_D, PGDH, PPS, THD2 and/or GLUDy367 7 6.29139 0.03885 ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, PPCK,THD2 and/or GLUDy 368 7 6.27848 0.32143 ADHEr, HEX1, LDH_D, NADH12,NADH6, PPS, THD2 and/or GLUDy 369 7 6.27504 0.1561 ADHEr, ATPS4r, FUM,LDH_D, NADH6, PGI, TAL 370 7 6.19234 0.13601 ADHEr, EDA and/or PGDHY,LDH_D, MDH, NADH6, PYK, THD2 and/or GLUDy 371 7 6.19234 0.13601 ADHEr,LDH_D, MDH, NADH6, PGL and/or G6PDHy, PYK, THD2 and/or GLUDy 372 76.18835 0.15484 ADHEr, LDH_D, MDH, NADH6, PYK, RPE, THD2 and/or GLUDy373 7 6.16073 0.14508 ADHEr, EDA and/or PGDHY, LDH_D, MDH, NADH6, PPCK,PYK 374 7 6.16073 0.14508 ADHEr, LDH_D, MDH, NADH6, PGL and/or G6PDHy,PPCK, PYK 375 7 6.15763 0.16479 ADHEr, FUM, LDH_D, NADH6, PPCK, PYK, RPE376 7 6.15763 0.16479 ADHEr, LDH_D, MDH, NADH6, PPCK, PYK, RPE 377 76.14691 0.14904 ADHEr, GLCpts, LDH_D, MDH, NADH12, NADH6, PPCK 378 76.14691 0.14904 ADHEr, FUM, GLCpts, LDH_D, NADH12, NADH6, PPCK 379 76.14046 0.17035 ADHEr, GLCpts, LDH_D, NADH6, PPCK, RPE, THD2 and/orGLUDy 380 7 6.13285 0.17281 ADHEr, LDH_D, MDH, NADH12, NADH6, RPE, THD2and/or GLUDy 381 7 6.09969 0.18355 ADHEr, FUM, LDH_D, NADH12, NADH6,PPCK, RPE 382 7 6.09969 0.18355 ADHEr, LDH_D, MDH, NADH12, NADH6, PPCK,RPE 383 7 6.07495 0.19156 ADHEr, FUM, GLCpts, LDH_D, NADH6, RPE, THD2and/or GLUDy 384 7 6.02503 0.20772 ADHEr, FUM, LDH_D, NADH12, NADH6,RPE, THD2 and/or GLUDy 385 7 5.9952 0.19254 ADHEr, FUM, HEX1, LDH_D,NADH12, NADH6, THD2 and/or GLUDy 386 7 5.9868 0.32178 ADHEr, ATPS4r,HEX1, LDH_D, NADH6, PPS, THD2 and/or GLUDy 387 7 5.98012 0.20938 ADHEr,FUM, LDH_D, NADH12, NADH6, TAL, THD2 and/or GLUDy 388 7 5.94711 0.3261ADHEr, HEX1, ICL, LDH_D, PPS, TAL, THD2 and/or GLUDy 389 7 5.947110.3261 ADHEr, HEX1, LDH_D, MALS, PPS, TAL, THD2 and/or GLUDy 390 75.93766 0.22464 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, NADH12,THD2 and/or GLUDy 391 7 5.84213 0.21172 ACKr and/or PTAr, ADHEr, HEX1,LDH_D, NADH12, NADH6, THD2 and/or GLUDy 392 7 5.77732 0.09217 ACKrand/or PTAr, ADHEr, ATPS4r, LDH_D, PGDH, PPCK, THD2 and/or GLUDy 393 75.77732 0.09217 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PGL and/orG6PDHy, PPCK, THD2 and/or GLUDy 394 7 5.7407 0.18622 ACKr and/or PTAr,ADHEr, GLUDy, HEX1, LDH_D, NADH6, THD2 and/or GLUDy 395 7 5.726080.09261 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, TAL, THD2 and/orGLUDy 396 7 5.67891 0.09302 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D,PPCK, RPE, THD2 and/or GLUDy 397 7 5.62569 0.20722 ACKr and/or PTAr,ADHEr, HEX1, LDH_D, NADH6, TAL, THD2 and/or GLUDy 398 7 5.60804 0.32285ADHEr, GLU5K, HEX1, LDH_D, NADH6, TAL, THD2 and/or GLUDy 399 7 5.608040.32285 ADHEr, G5SD, HEX1, LDH_D, NADH6, TAL, THD2 and/or GLUDy 400 75.60791 0.32289 ADHEr, ASNS2, HEX1, LDH_D, NADH6, TAL, THD2 and/or GLUDy401 7 5.59783 0.33545 ADHEr, HEX1, ICL, LDH_D, PPS, RPE, THD2 and/orGLUDy 402 7 5.59783 0.33545 ADHEr, HEX1, LDH_D, MALS, PPS, RPE, THD2and/or GLUDy 403 7 5.58104 0.23062 ACKr and/or PTAr, ADHEr, ATPS4r,HEX1, LDH_D, PGL and/or G6PDHy, THD2 and/or GLUDy 404 7 5.58104 0.23062ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, PGDH, THD2 and/or GLUDy405 7 5.35846 0.23124 ACKr and/or PTAr, ADHEr, ATPS4r, HEX1, LDH_D, TAL,THD2 and/or GLUDy 406 7 5.27565 0.07793 ADHEr, EDA and/or PGDHY, LDH_D,MDH, PPCK, PYK, THD2 and/or GLUDy 407 7 5.19178 0.1965 ADHEr, FUM,LDH_D, PFLi, PGDH, PGI, TAL 408 7 5.19178 0.1965 ADHEr, LDH_D, MDH,PFLi, PGDH, PGI, TAL 409 7 5.16412 0.3061 ADHEr, HEX1, LDH_D, PGL and/orG6PDHy, PPS, TAL, THD2 and/or GLUDy 410 7 5.16412 0.3061 ADHEr, HEX1,LDH_D, PGDH, PPS, TAL, THD2 and/or GLUDy 411 7 5.15646 0.16031 ADHEr,ATPS4r, LDH_D, PFLi, PGDH, PGI, TAL 412 7 5.14711 0.23182 ACKr and/orPTAr, ADHEr, ATPS4r, HEX1, LDH_D, RPE, THD2 and/or GLUDy 413 7 5.065930.18784 ADHEr, LDH_D, PFLi, PGDH, PGI, PPS, THD2 and/or GLUDy 414 75.06354 0.16677 ADHEr, ATPS4r, GLYCL, LDH_D, PFLi, PGDH, PGI 415 75.04267 0.18882 ADHEr, LDH_D, PFLi, PGI, PPS, TAL, THD2 and/or GLUDy 4167 5.02125 0.18972 ADHEr, LDH_D, PFLi, PGI, PPS, RPE, THD2 and/or GLUDy417 7 4.56087 0.18955 ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, RPE,THD2 and/or GLUDy 418 7 4.53442 0.18932 ACKr and/or PTAr, ADHEr, FUM,HEX1, LDH_D, TAL, THD2 and/or GLUDy 419 7 4.50549 0.18907 ACKr and/orPTAr, ADHEr, FUM, HEX1, LDH_D, PGL and/or G6PDHy, THD2 and/or GLUDy 4207 4.50549 0.18907 ACKr and/or PTAr, ADHEr, FUM, HEX1, LDH_D, PGDH, THD2and/or GLUDy 421 7 4.43945 0.24195 ADHEr, FUM, GLUDy, HEX1, LDH_D, RPE,THD2 and/or GLUDy 422 7 4.33649 0.2698 ADHEr, ASNS2, FUM, G5SD, LDH_D,TAL, THD2 and/or GLUDy 423 7 4.33649 0.2698 ADHEr, ASNS2, FUM, GLU5K,LDH_D, TAL, THD2 and/or GLUDy 424 7 3.82299 0.23376 ACKr and/or PTAr,ADHEr, CITL, HEX1, LDH_D, PPS, THD2 and/or GLUDy 425 7 3.40957 0.10927ADHEr, FRD and/or SUCD4, LDH_D, PGDH, PPS, TAL, THD2 and/or GLUDy 426 73.40957 0.10927 ADHEr, FRD and/or SUCD4, LDH_D, PGL and/or G6PDHy, PPS,TAL, THD2 and/or GLUDy 427 7 3.30702 0.26731 ACKr and/or PTAr, ADHEr,ATPS4r, FUM, LDH_D, TAL, THD2 and/or GLUDy 428 7 1.7546 0.28013 ACKrand/or PTAr, ADHEr, CITL, FUM, LDH_D, PPS, THD2 and/or GLUDy 429 70.1401 0.25687 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, RPE, SUCOAS430 7 0.13962 0.25599 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH,SUCOAS, TAL 431 7 0.13909 0.25503 ACKr and/or PTAr, ADHEr, GLCpts,LDH_D, MDH, PGDH, SUCOAS 432 7 0.13909 0.25503 ACKr and/or PTAr, ADHEr,GLCpts, LDH_D, MDH, PGL and/or G6PDHy, SUCOAS 433 3 0.3313622840.248770484 ADHEr, LDH_D, PFK and/or FBA and/or TPI 434 3 4.18869160.310192487 ADHEr, FRD and/or SUCD4, LDH_D 435 4 2.773932742 0.386261ADHEr, ASNS2, ATPS4r, LDH_D 436 4 2.792658751 0.384018912 ADHEr, ATPS4r,CBMK2, LDH_D 437 4 5.490131382 0.33737393 ADHEr, CBMK2, LDH_D, NADH6 4384 6.078666008 0.168610416 ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI439 4 0.474685279 0.246014656 ADHEr, LDH_D, PFK and/or FBA and/or TPI,RPE 440 4 0.406720198 0.247321495 ADHEr, LDH_D, PFK and/or FBA and/orTPI, TAL 441 4 0.406720198 0.247321495 ADHEr, LDH_D, PFK and/or FBAand/or TPI, TKT1 442 4 0.541935885 0.244721556 ADHEr, LDH_D, PFK and/orFBA and/or TPI, TKT2 443 4 4.227003016 0.295544651 ADHEr, FRD and/orSUCD4, FUM, LDH_D 444 4 2.774411941 0.386203624 ADHEr, ATPS4r, G5SD,LDH_D 445 4 2.774411941 0.386203624 ADHEr, ATPS4r, GLU5K, LDH_D 446 42.552977098 0.152232967 ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI447 4 5.500068328 0.33452449 ADHEr, HEX1, LDH_D, NADH6 448 4 4.2263622190.295789651 ADHEr, FRD and/or SUCD4, HEX1, LDH_D 449 4 0.9993587090.388175844 ADHEr, HEX1, LDH_D, TKT1 450 4 1.300899111 0.38573732 ADHEr,HEX1, LDH_D, TKT2 451 4 4.374206544 0.239263432 ADHEr, FRD and/or SUCD4,LDH_D, MDH 452 4 4.29486095 0.269600115 ADHEr, FRD and/or SUCD4, LDH_D,ME2 453 4 4.488943189 0.195395474 ADHEr, FRD and/or SUCD4, LDH_D, PPCK454 4 4.312306765 0.279635371 ADHEr, LDH_D, PPCK, TAL 455 4 4.3123067650.279635371 ADHEr, LDH_D, PPCK, TKT1 456 4 4.394666553 0.278410255ADHEr, LDH_D, PPCK, TKT2 457 4 4.290442023 0.306273023 ADHEr, FRD and/orSUCD4, LDH_D, RPE 458 4 4.242231032 0.308130129 ADHEr, FRD and/or SUCD4,LDH_D, TAL 459 4 3.02411229 0.384504476 ADHEr, ATPS4r, LDH_D, TKT1 460 45.556064858 0.338701395 ADHEr, LDH_D, NADH6, TKT1 461 4 4.2422310320.308130129 ADHEr, FRD and/or SUCD4, LDH_D, TKT1 462 4 3.4917388830.378757536 ADHEr, ATPS4r, LDH_D, TKT2 463 4 5.694356782 0.335394947ADHEr, LDH_D, NADH6, TKT2 464 4 4.338075359 0.30443817 ADHEr, FRD and/orSUCD4, LDH_D, TKT2 465 5 3.269904021 0.380186443 ADHEr, ASNS2, ATPS4r,LDH_D, RPE 466 5 3.035292503 0.383059918 ADHEr, ASNS2, ATPS4r, LDH_D,TAL 467 5 2.786583612 0.384746295 ADHEr, ASNS2, ATPS4r, GLU5K, LDH_D 4685 3.035292503 0.383059918 ADHEr, ASNS2, ATPS4r, LDH_D, TKT1 469 54.891772757 0.345085032 ADHEr, ATPS4r, LDH_D, ME2, THD2 and/or GLUDy 4705 4.906959994 0.170872463 ADHEr, ATPS4r, LDH_D, PFK and/or FBA and/orTPI, PFLi 471 5 2.80469336 0.382577993 ADHEr, ASNS2, ATPS4r, CBMK2,LDH_D 472 5 4.199370617 0.306109514 ADHEr, ASNS2, CBMK2, FRD and/orSUCD4, LDH_D 473 5 3.285503322 0.378014123 ADHEr, ATPS4r, CBMK2, LDH_D,RPE 474 5 3.052359629 0.380854732 ADHEr, ATPS4r, CBMK2, LDH_D, TAL 475 52.805163465 0.382521707 ADHEr, ATPS4r, CBMK2, G5SD, LDH_D 476 54.199487305 0.3060649 ADHEr, CBMK2, FRD and/or SUCD4, G5SD, LDH_D 477 52.805163465 0.382521707 ADHEr, ATPS4r, CBMK2, GLU5K, LDH_D 478 54.199487305 0.3060649 ADHEr, CBMK2, FRD and/or SUCD4, GLU5K, LDH_D 479 55.566341358 0.335567361 ADHEr, CBMK2, LDH_D, NADH6, TAL 480 56.853869476 0.27580388 ADHEr, CBMK2, LDH_D, NADH6, PFLi 481 53.052359629 0.380854732 ADHEr, ATPS4r, CBMK2, LDH_D, TKT1 482 55.566341358 0.335567361 ADHEr, CBMK2, LDH_D, NADH6, TKT1 483 53.515194954 0.375215574 ADHEr, ATPS4r, CBMK2, LDH_D, TKT2 484 54.886253722 0.208900511 ADHEr, LDH_D, MDH, PFK and/or FBA and/or TPI,PFLi 485 5 4.621960711 0.144538077 ADHEr, FRD and/or SUCD4, LDH_D, MDH,PFK and/or FBA and/or TPI 486 5 6.151957387 0.166626529 ADHEr, LDH_D,NADH6, PFK and/or FBA and/or TPI, RPE 487 5 6.117214665 0.167566962ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI, TAL 488 5 0.4363762980.238196669 ADHEr, LDH_D, PFK and/or FBA and/or TPI, PGDH, TAL 489 56.117214665 0.167566962 ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI,TKT1 490 5 0.436376298 0.238196669 ADHEr, LDH_D, PFK and/or FBA and/orTPI, PGDH, TKT1 491 5 6.186312313 0.165696592 ADHEr, LDH_D, NADH6, PFKand/or FBA and/or TPI, TKT2 492 5 5.556162772 0.318439293 ADHEr, CBMK2,FUM, LDH_D, NADH6 493 5 4.886253722 0.208900511 ADHEr, FUM, LDH_D, PFKand/or FBA and/or TPI, PFLi 494 5 4.613740943 0.14768079 ADHEr, FRDand/or SUCD4, FUM, LDH_D, PFK and/or FBA and/or TPI 495 5 6.0881951550.307660476 ADHEr, FUM, LDH_D, ME2, THD2 and/or GLUDy 496 5 5.6840649390.281763084 ADHEr, FUM, LDH_D, ME2, NADH6 497 5 4.673184878 0.145745102ADHEr, FRD and/or SUCD4, FUM, LDH_D, PGI 498 5 4.572772914 0.163344346ADHEr, FRD and/or SUCD4, FUM, LDH_D, PPCK 499 5 4.382533025 0.251079405ADHEr, FUM, LDH_D, PPCK, TAL 500 5 4.382533025 0.251079405 ADHEr, FUM,LDH_D, PPCK, TKT1 501 5 4.457618973 0.24945663 ADHEr, FUM, LDH_D, PPCK,TKT2 502 5 4.374206544 0.239263432 ADHEr, FRD and/or SUCD4, FUM, LDH_D,ME2 503 5 4.324805294 0.29144049 ADHEr, FRD and/or SUCD4, FUM, LDH_D,RPE 504 5 4.278495915 0.29338381 ADHEr, FRD and/or SUCD4, FUM, LDH_D,TAL 505 5 5.619129051 0.319468644 ADHEr, FUM, LDH_D, NADH6, TKT1 506 51.418161594 0.362515745 ADHEr, FUM, LDH_D, PGDH, TKT1 507 5 1.4181615940.362515745 ADHEr, FUM, LDH_D, PGL and/or G6PDHy, TKT1 508 5 4.2784959150.29338381 ADHEr, FRD and/or SUCD4, FUM, LDH_D, TKT1 509 5 5.7509647160.315868237 ADHEr, FUM, LDH_D, NADH6, TKT2 510 5 4.370505222 0.289522745ADHEr, FRD and/or SUCD4, FUM, LDH_D, TKT2 511 5 2.786583612 0.384746295ADHEr, ASNS2, ATPS4r, G5SD, LDH_D 512 5 3.270303172 0.380130859 ADHEr,ATPS4r, G5SD, LDH_D, RPE 513 5 3.035729231 0.38300349 ADHEr, ATPS4r,G5SD, LDH_D, TAL 514 5 3.035729231 0.38300349 ADHEr, ATPS4r, G5SD,LDH_D, TKT1 515 5 3.270303172 0.380130859 ADHEr, ATPS4r, GLU5K, LDH_D,RPE 516 5 3.035729231 0.38300349 ADHEr, ATPS4r, GLU5K, LDH_D, TAL 517 54.101998016 0.149473222 ADHEr, ATPS4r, HEX1, LDH_D, PFK and/or FBAand/or TPI 518 5 5.510369128 0.331570715 ADHEr, CBMK2, HEX1, LDH_D,NADH6 519 5 3.359019108 0.150796918 ADHEr, HEX1, LDH_D, MDH, PFK and/orFBA and/or TPI 520 5 4.301255739 0.267155156 ADHEr, FRD and/or SUCD4,FUM, HEX1, LDH_D 521 5 4.146125845 0.347209192 ADHEr, FUM, HEX1, LDH_D,TKT1 522 5 4.24954345 0.345156537 ADHEr, FUM, HEX1, LDH_D, TKT2 523 55.64062493 0.332159859 ADHEr, HEX1, LDH_D, NADH6, RPE 524 5 5.5738298930.333283574 ADHEr, HEX1, LDH_D, NADH6, TAL 525 5 6.871954799 0.271451843ADHEr, HEX1, LDH_D, NADH6, PFLi 526 5 1.026318237 0.371585169 ADHEr,HEX1, LDH_D, PGDH, TAL 527 5 1.026318237 0.371585169 ADHEr, HEX1, LDH_D,PGL and/or G6PDHy, TAL 528 5 4.321580803 0.292832307 ADHEr, FRD and/orSUCD4, HEX1, LDH_D, RPE 529 5 4.27640137 0.294235511 ADHEr, FRD and/orSUCD4, HEX1, LDH_D, TAL 530 5 4.292100506 0.270655532 ADHEr, FRD and/orSUCD4, HEX1, LDH_D, THD2 and/or GLUDy 531 5 5.573829893 0.333283574ADHEr, HEX1, LDH_D, NADH6, TKT1 532 5 1.026318237 0.371585169 ADHEr,HEX1, LDH_D, PGDH, TKT1 533 5 1.026318237 0.371585169 ADHEr, HEX1,LDH_D, PGL and/or G6PDHy, TKT1 534 5 4.27640137 0.294235511 ADHEr, FRDand/or SUCD4, HEX1, LDH_D, TKT1 535 5 4.093661719 0.368542577 ADHEr,HEX1, LDH_D, THD2 and/or GLUDy, TKT1 536 5 5.706971061 0.331043695ADHEr, HEX1, LDH_D, NADH6, TKT2 537 5 4.366331362 0.291442423 ADHEr, FRDand/or SUCD4, HEX1, LDH_D, TKT2 538 5 4.197378033 0.36914891 ADHEr,HEX1, LDH_D, THD2 and/or GLUDy, TKT2 539 5 6.584217406 0.204283888ADHEr, ASPT, CBMK2, LDH_D, MDH 540 5 4.679174494 0.143073998 ADHEr, FRDand/or SUCD4, LDH_D, MDH, PGI 541 5 4.572772914 0.163344346 ADHEr, FRDand/or SUCD4, LDH_D, MDH, PPCK 542 5 4.382533025 0.251079405 ADHEr,LDH_D, MDH, PPCK, TAL 543 5 4.382533025 0.251079405 ADHEr, LDH_D, MDH,PPCK, TKT1 544 5 4.457618973 0.24945663 ADHEr, LDH_D, MDH, PPCK, TKT2545 5 4.450595421 0.237144547 ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE546 5 4.414328365 0.238150528 ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL547 5 6.807575806 0.294019202 ADHEr, ATPS4r, LDH_D, MDH, TKT1 548 55.745562276 0.280910173 ADHEr, LDH_D, MDH, NADH6, TKT1 549 5 4.4143283650.238150528 ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT1 550 5 6.4557453410.299423269 ADHEr, ATPS4r, LDH_D, MDH, TKT2 551 5 5.856779801 0.27936769ADHEr, LDH_D, MDH, NADH6, TKT2 552 5 4.486557371 0.23614703 ADHEr, FRDand/or SUCD4, LDH_D, MDH, TKT2 553 5 6.254106519 0.315300181 ADHEr,LDH_D, ME2, NADH6, THD2 and/or GLUDy 554 5 4.339242812 0.268682419ADHEr, FRD and/or SUCD4, LDH_D, ME2, TAL 555 5 6.224794308 0.229694348ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or GLUDy 556 5 6.8763531640.277060587 ADHEr, LDH_D, NADH6, PFLi, TAL 557 5 7.122630133 0.211129547ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi 558 5 4.216706856 0.262624991ADHEr, FRD and/or SUCD4, LDH_D, PFLi, THD2 and/or GLUDy 559 55.563197905 0.034931763 ADHEr, EDA and/or PGDHY, FRD and/or SUCD4,LDH_D, PGI 560 5 5.66839105 0.263566425 ADHEr, LDH_D, ME2, PGL and/orG6PDHy, THD2 and/or GLUDy 561 5 4.552158784 0.193305823 ADHEr, FRDand/or SUCD4, LDH_D, PPCK, RPE 562 5 4.522175259 0.194296957 ADHEr, FRDand/or SUCD4, LDH_D, PPCK, TAL 563 5 5.924716044 0.226273464 ADHEr,LDH_D, NADH6, PPCK, TKT1 564 5 5.265518204 0.241270472 ADHEr, LDH_D,PFLi, PPCK, TKT1 565 5 4.522175259 0.194296957 ADHEr, FRD and/or SUCD4,LDH_D, PPCK, TKT1 566 5 6.01594542 0.224464061 ADHEr, LDH_D, NADH6,PPCK, TKT2 567 5 5.456414614 0.240407987 ADHEr, LDH_D, PFLi, PPCK, TKT2568 5 4.581837961 0.192324751 ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TKT2569 5 3.352437372 0.115790546 ADHEr, FADH4, LDH_D, PPS, THD2 and/orGLUDy 570 5 4.568751314 0.164881948 ADHEr, FRD and/or SUCD4, LDH_D,PPCK, PYK 571 5 4.379456454 0.26785091 ADHEr, FRD and/or SUCD4, LDH_D,ME2, RPE 572 5 3.035729231 0.38300349 ADHEr, ATPS4r, GLU5K, LDH_D, TKT1573 5 6.876353164 0.277060587 ADHEr, LDH_D, NADH6, PFLi, TKT1 574 54.339242812 0.268682419 ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT1 575 54.419421961 0.267024532 ADHEr, FRD and/or SUCD4, LDH_D, ME2, TKT2 576 56.185324416 0.166037364 ADHEr, LDH_D, NADH6, PGI, TKT2 577 5 6.9368719030.274483161 ADHEr, LDH_D, NADH6, PFLi, TKT2 578 6 7.6146344120.238178822 ADHEr, ATPS4r, CBMK2, LDH_D, MDH, NADH6 579 6 7.6036793010.239793337 ADHEr, ATPS4r, FUM, LDH_D, ME2, NADH6 580 6 7.2691540450.188693079 ADHEr, FUM, LDH_D, PFLi, PPCK, TKT2 581 6 7.5391726550.163831584 ADHEr, ASNS2, CBMK2, HEX1, LDH_D, PGI 582 6 7.5393127320.163809352 ADHEr, CBMK2, G5SD, HEX1, LDH_D, PGI 583 6 7.5393127320.163809352 ADHEr, CBMK2, GLU5K, HEX1, LDH_D, PGI 584 6 7.4978750690.097945448 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MDH, THD2 and/or GLUDy585 6 7.316598461 0.164453176 ADHEr, FRD and/or SUCD4, HEX1, LDH_D,PFLi, THD2 and/or GLUDy 586 6 7.269154045 0.188693079 ADHEr, LDH_D, MDH,PFLi, PPCK, TKT2 587 6 7.389036066 0.088156259 ADHEr, ASPT, FRD and/orSUCD4, LDH_D, MDH, THD2 and/or GLUDy 588 6 8.258765764 0.152284822ADHEr, FRD and/or SUCD4, LDH_D, ME2, PFLi, THD2 and/or GLUDy 589 68.999143093 0.036289126 ADHEr, FRD and/or SUCD4, LDH_D, PFLi, PPCK, THD2and/or GLUDy

TABLE 16 Knockout strain designs for increased production of HMDA,showing yields of HMDA and biomass. New Num HMDA. Design ID KO YieldBiomass Rxnlist 1 3 2.32302 0.35737 ADHEr, LDH_D, NADH6 2 3 2.128640.28397 ADHEr, LDH_D, PPCK 3 3 1.2553 0.38773 ADHEr, ATPS4r, LDH_D 4 31.11254 0.26898 ADHEr, FRD and/or SUCD4, LDH_D 5 3 0.67068 0.37924ADHEr, FUM, LDH_D 6 3 0.57155 0.26988 ADHEr, LDH_D, PGI 7 3 0.277470.38953 ADHEr, HEX1, LDH_D 8 4 4.46145 0.20591 ADHEr, FRD and/or SUCD4,LDH_D, MDH 9 4 4.38802 0.23398 ADHEr, FRD and/or SUCD4, HEX1, LDH_D 10 44.03852 0.23697 ADHEr, HEX1, LDH_D, PGI 11 4 3.73656 0.30662 ADHEr,LDH_D, NADH6, PFLi 12 4 3.05653 0.20692 ACKr and/or PTAr, ADHEr, LDH_D,PPCK 13 4 2.59406 0.20532 ADHEr, LDH_D, NADH6, PGI 14 4 2.58406 0.27187ADHEr, ASPT, LDH_D, MDH 15 4 2.46512 0.26229 ADHEr, LDH_D, NADH6, PPCK16 4 2.41847 0.29351 ADHEr, LDH_D, MDH, NADH6 17 4 2.38828 0.35417ADHEr, LDH_D, NADH6, RPE 18 4 2.29878 0.17947 ADHEr, LDH_D, PGI, PPCK 194 2.17588 0.24785 ADHEr, FUM, LDH_D, THD2 and/or GLUDy 20 4 2.147450.26084 ADHEr, EDA and/or PGDHY, LDH_D, PGI 21 4 2.03987 0.35185 ADHEr,FUM, HEX1, LDH_D 22 4 1.48168 0.38161 ADHEr, ATPS4r, LDH_D, RPE 23 41.47082 0.28924 ADHEr, LDH_D, MDH, THD2 and/or GLUDy 24 4 1.3746 0.3845ADHEr, ATPS4r, LDH_D, TAL 25 4 1.26025 0.2616 ADHEr, FRD and/or SUCD4,LDH_D, SUCOAS 26 4 0.939 0.26603 ADHEr, LDH_D, PFLi, PGI 27 4 0.761490.2867 ADHEr, ASPT, FUM, LDH_D 28 4 0.38353 0.38695 ADHEr, HEX1, LDH_D,RPE 29 4 0.33312 0.38818 ADHEr, HEX1, LDH_D, TAL 30 5 5.53962 0.16718ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, MDH 31 5 5.40224 0.22385 ADHEr,HEX1, LDH_D, PFLi, PGI 32 5 5.13405 0.1802 ADHEr, ASPT, LDH_D, MDH, PFLi33 5 5.0304 0.08351 ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PPCK 34 54.96585 0.07704 ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, THD2 and/orGLUDy 35 5 4.88127 0.20295 ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PGI 365 4.78114 0.2037 ADHEr, HEX1, LDH_D, NADH6, PGI 37 5 4.75518 0.09361ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK 38 5 4.61203 0.20383 ACKrand/or PTAr, ADHEr, LDH_D, NADH6, PGI 39 5 4.60391 0.15144 ADHEr, FRDand/or SUCD4, LDH_D, MDH, THD2 and/or GLUDy 40 5 4.56119 0.16777 ADHEr,FRD and/or SUCD4, GLCpts, LDH_D, MDH 41 5 4.52834 0.20359 ADHEr, FRDand/or SUCD4, LDH_D, MDH, RPE 42 5 4.51845 0.20259 ADHEr, FRD and/orSUCD4, LDH_D, MDH, SUCOAS 43 5 4.46585 0.23056 ADHEr, FRD and/or SUCD4,HEX1, LDH_D, RPE 44 5 4.45522 0.22919 ADHEr, FRD and/or SUCD4, HEX1,LDH_D, SUCOAS 45 5 4.44183 0.21341 ADHEr, FRD and/or SUCD4, HEX1, LDH_D,THD2 and/or GLUDy 46 5 4.44155 0.21352 ADHEr, ATPS4r, FRD and/or SUCD4,HEX1, LDH_D 47 5 4.42059 0.20565 ADHEr, ASPT, LDH_D, MDH, SUCOAS 48 54.36907 0.15859 ADHEr, EDA and/or PGDHY, LDH_D, PGI, PPCK 49 5 4.360670.16481 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, PGI 50 5 4.33827 0.24026ADHEr, LDH_D, MDH, NADH6, THD2 and/or GLUDy 51 5 4.24585 0.21569 ADHEr,ASPT, FUM, LDH_D, MDH 52 5 4.24585 0.21569 ADHEr, ASPT, ICL, LDH_D, MDH53 5 4.24585 0.21569 ADHEr, ASPT, LDH_D, MALS, MDH 54 5 4.21843 0.08488ADHEr, FRD and/or SUCD4, LDH_D, PPCK, THD2 and/or GLUDy 55 5 4.182650.21782 ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH 56 5 4.13301 0.24796ADHEr, ASPT, FUM, HEX1, LDH_D 57 5 4.03737 0.23515 ACKr and/or PTAr,ADHEr, EDA and/or PGDHY, LDH_D, PGI 58 5 4.02056 0.22023 ADHEr, ASPT,LDH_D, MDH, THD2 and/or GLUDy 59 5 4.0188 0.18437 ADHEr, LDH_D, NADH6,PFLi, PPCK 60 5 3.96576 0.20734 ADHEr, FUM, LDH_D, PFLi, PPCK 61 53.96576 0.20734 ADHEr, LDH_D, MDH, PFLi, PPCK 62 5 3.96266 0.23773 ACKrand/or PTAr, ADHEr, LDH_D, MDH, NADH6 63 5 3.92623 0.22446 ADHEr, LDH_D,MDH, NADH6, PFLi 64 5 3.81332 0.27196 ACKr and/or PTAr, ADHEr, FUM,HEX1, LDH_D 65 5 3.81332 0.27196 ACKr and/or PTAr, ADHEr, HEX1, LDH_D,MDH 66 5 3.79273 0.28229 ADHEr, HEX1, LDH_D, NADH6, PFLi 67 5 3.776860.28031 ACKr and/or PTAr, ADHEr, HEX1, LDH_D, NADH6 68 5 3.74717 0.3358ADHEr, FUM, LDH_D, PFLi, THD2 and/or GLUDy 69 5 3.69789 0.22154 ADHEr,LDH_D, NADH6, PPCK, THD2 and/or GLUDy 70 5 3.69693 0.23186 ADHEr, ASPT,ATPS4r, LDH_D, MDH 71 5 3.57925 0.1851 ACKr and/or PTAr, ADHEr, LDH_D,NADH6, PPCK 72 5 3.55622 0.22164 ADHEr, FUM, LDH_D, PPCK, THD2 and/orGLUDy 73 5 3.55622 0.22164 ADHEr, LDH_D, MDH, PPCK, THD2 and/or GLUDy 745 3.51036 0.26322 ADHEr, ATPS4r, LDH_D, MDH, NADH6 75 5 3.44912 0.18587ACKr and/or PTAr, ADHEr, LDH_D, MDH, PPCK 76 5 3.44912 0.18587 ACKrand/or PTAr, ADHEr, FUM, LDH_D, PPCK 77 5 3.23914 0.31877 ADHEr, ATPS4r,FDH2, LDH_D, NADH6 78 5 3.23669 0.2512 ADHEr, EDA and/or PGDHY, LDH_D,PFLi, PGI 79 5 3.22027 0.23733 ADHEr, ATPS4r, LDH_D, NADH6, PPCK 80 53.21813 0.22145 ADHEr, ATPS4r, LDH_D, PFLi, PPCK 81 5 3.20482 0.28717ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, SUCOAS 82 5 3.19987 0.14763ADHEr, LDH_D, PFLi, PGI, PPCK 83 5 3.04087 0.23963 ADHEr, ATPS4r, FUM,LDH_D, PPCK 84 5 3.04087 0.23963 ADHEr, ATPS4r, LDH_D, MDH, PPCK 85 53.01019 0.28849 ADHEr, HEX1, LDH_D, MDH, THD2 and/or GLUDy 86 5 2.941610.20506 ADHEr, ATPS4r, LDH_D, NADH6, PGI 87 5 2.88011 0.33864 ADHEr,ATPS4r, FUM, LDH_D, NADH6 88 5 2.85293 0.25411 ADHEr, ASPT, LDH_D, MDH,NADH6 89 5 2.75034 0.26847 ADHEr, LDH_D, MDH, PFLi, THD2 and/or GLUDy 905 2.74759 0.33105 ADHEr, ATPS4r, HEX1, LDH_D, NADH6 91 5 2.67997 0.17637ADHEr, ATPS4r, LDH_D, PGI, PPCK 92 5 2.67853 0.21976 ADHEr, ASPT, LDH_D,MDH, PYK 93 5 2.65374 0.15874 ADHEr, LDH_D, NADH6, PGI, PPCK 94 52.65207 0.23435 ADHEr, ASPT, GLCpts, LDH_D, MDH 95 5 2.64298 0.23937ADHEr, ASPT, LDH_D, MDH, PPCK 96 5 2.63715 0.26997 ADHEr, ASPT, LDH_D,MDH, RPE 97 5 2.60095 0.33049 ADHEr, ATPS4r, FUM, HEX1, LDH_D 98 52.52584 0.22167 ADHEr, LDH_D, NADH6, PPCK, PYK 99 5 2.5171 0.22752ADHEr, GLCpts, LDH_D, NADH6, PPCK 100 5 2.48801 0.24981 ADHEr, ATPS4r,LDH_D, MDH, THD2 and/or GLUDy 101 5 2.47253 0.25734 ADHEr, GLCpts,LDH_D, MDH, NADH6 102 5 2.47049 0.29207 ADHEr, LDH_D, MDH, NADH6, RPE103 5 2.38845 0.0995 ADHEr, LDH_D, PGI, PPCK, THD2 and/or GLUDy 104 52.3823 0.10498 ADHEr, FUM, LDH_D, PGI, THD2 and/or GLUDy 105 5 2.38230.10498 ADHEr, LDH_D, MDH, PGI, THD2 and/or GLUDy 106 5 2.37356 0.34412ADHEr, HEX1, LDH_D, NADH6, TAL 107 5 2.36643 0.29114 ADHEr, ATPS4r,LDH_D, MDH, PGDH 108 5 2.33103 0.23832 ADHEr, ATPS4r, LDH_D, PPCK, THD2and/or GLUDy 109 5 2.32447 0.13422 ADHEr, GLUDy, LDH_D, MDH, THD2 and/orGLUDy 110 5 2.29184 0.29402 ADHEr, ATPS4r, LDH_D, MDH, TAL 111 5 2.2820.28884 ACKr and/or PTAr, ADHEr, LDH_D, MDH, THD2 and/or GLUDy 112 52.22251 0.2967 ADHEr, ATPS4r, LDH_D, MDH, RPE 113 5 2.22197 0.24001ADHEr, LDH_D, MDH, RPE, THD2 and/or GLUDy 114 5 2.21206 0.24857 ADHEr,GLCpts, LDH_D, PPCK, RPE 115 5 2.20561 0.23941 ADHEr, LDH_D, MDH, TAL,THD2 and/or GLUDy 116 5 2.20149 0.22826 ADHEr, FUM, LDH_D, PPCK, PYK 1175 2.20149 0.22826 ADHEr, LDH_D, MDH, PPCK, PYK 118 5 2.19372 0.24909ADHEr, GLCpts, LDH_D, PPCK, TAL 119 5 2.19132 0.23604 ADHEr, GLCpts,LDH_D, MDH, PPCK 120 5 2.19132 0.23604 ADHEr, FUM, GLCpts, LDH_D, PPCK121 5 2.18792 0.26941 ADHEr, FUM, LDH_D, PPCK, RPE 122 5 2.18792 0.26941ADHEr, LDH_D, MDH, PPCK, RPE 123 5 2.18777 0.23875 ADHEr, LDH_D, MDH,PGDH, THD2 and/or GLUDy 124 5 2.09486 0.34975 ADHEr, FUM, HEX1, LDH_D,RPE 125 5 1.89618 0.34765 ADHEr, ATPS4r, HEX1, LDH_D, THD2 and/or GLUDy126 5 1.74629 0.36027 ADHEr, HEX1, LDH_D, PFLi, PPS 127 5 1.73743 0.3202ADHEr, ATPS4r, HEX1, LDH_D, PFLi 128 5 1.73543 0.31113 ADHEr, HEX1,LDH_D, MDH, PFLi 129 5 1.69405 0.23402 ADHEr, LDH_D, PFLi, PGDH, PGI 1305 1.68439 0.23524 ADHEr, LDH_D, PFLi, PGI, TAL 131 5 1.67549 0.23637ADHEr, LDH_D, PFLi, PGI, RPE 132 5 1.66962 0.16853 ADHEr, ATPS4r, LDH_D,PFLi, PGI 133 5 1.66579 0.35584 ADHEr, HEX1, LDH_D, PPS, THD2 and/orGLUDy 134 5 1.44831 0.25358 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4,LDH_D, SUCOAS 135 5 1.40898 0.31979 ADHEr, ATPS4r, HEX1, LDH_D, MDH 1365 1.33078 0.25859 ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, SUCOAS 137 51.28594 0.26157 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, ICL, LDH_D138 5 1.28594 0.26157 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D,MALS 139 5 1.15083 0.26676 ADHEr, ASPT, FUM, LDH_D, SUCOAS 140 5 1.085560.26021 ACKr and/or PTAr, ADHEr, LDH_D, PFLi, PGI 141 5 1.08281 0.26394ADHEr, FUM, LDH_D, PFLi, PGI 142 5 0.81325 0.32358 ACKr and/or PTAr,ADHEr, HEX1, LDH_D, PPS 143 5 0.73764 0.26814 ADHEr, LDH_D, MDH, PGI,THD5 144 5 0.73764 0.26814 ADHEr, FUM, LDH_D, PGI, THD5 145 6 6.492040.11612 ADHEr, ASPT, LDH_D, MDH, PFLi, THD2 and/or GLUDy 146 6 6.189990.07445 ADHEr, ATPS4r, FRD and/or SUCD4, GLCpts, LDH_D, MDH 147 6 6.14140.1578 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGL and/or G6PDHy 148 6 5.808570.1099 ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PYK 149 6 5.629480.09192 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, PGI 150 6 5.60131 0.13875ADHEr, EDA and/or PGDHY, LDH_D, PFLi, PGI, PPCK 151 6 5.59693 0.15008ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MDH, THD2 and/or GLUDy 152 65.56181 0.13846 ADHEr, EDA and/or PGDHY, FRD and/or SUCD4, LDH_D, MDH,THD2 and/or GLUDy 153 6 5.52668 0.15619 ADHEr, EDA and/or PGDHY, LDH_D,NADH6, PFLi, PGI 154 6 5.52416 0.10076 ADHEr, ASPT, LDH_D, MDH, PFLi,PGI 155 6 5.44943 0.16764 ADHEr, ASPT, ATPS4r, LDH_D, MDH, SUCOAS 156 65.43906 0.17387 ADHEr, ASPT, ATPS4r, FUM, LDH_D, MDH 157 6 5.439060.17387 ADHEr, ASPT, ATPS4r, ICL, LDH_D, MDH 158 6 5.43906 0.17387ADHEr, ASPT, ATPS4r, LDH_D, MALS, MDH 159 6 5.4269 0.11474 ADHEr, ASPT,EDA and/or PGDHY, LDH_D, MDH, PGI 160 6 5.39974 0.15728 ADHEr, LDH_D,MDH, PFLi, PPCK, THD2 and/or GLUDy 161 6 5.39974 0.15728 ADHEr, FUM,LDH_D, PFLi, PPCK, THD2 and/or GLUDy 162 6 5.36516 0.17457 ADHEr, LDH_D,MDH, NADH6, PFLi, THD2 and/or GLUDy 163 6 5.29594 0.12992 ADHEr, ASPT,LDH_D, MDH, PFLi, PYK 164 6 5.28218 0.07163 ADHEr, ATPS4r, FRD and/orSUCD4, LDH_D, PGL and/or G6PDHy, PPCK 165 6 5.28218 0.07163 ADHEr,ATPS4r, FRD and/or SUCD4, LDH_D, PGDH, PPCK 166 6 5.27982 0.10569 ADHEr,ASPT, LDH_D, MDH, PGL and/or G6PDHy, PYK 167 6 5.27077 0.07217 ADHEr,ATPS4r, FRD and/or SUCD4, LDH_D, PPCK, TAL 168 6 5.26177 0.14053 ADHEr,ASPT, GLCpts, LDH_D, MDH, PFLi 169 6 5.26021 0.07267 ADHEr, ATPS4r, FRDand/or SUCD4, LDH_D, PPCK, RPE 170 6 5.22996 0.24218 ADHEr, HEX1, LDH_D,MDH, PFLi, THD2 and/or GLUDy 171 6 5.22996 0.24218 ADHEr, FUM, HEX1,LDH_D, PFLi, THD2 and/or GLUDy 172 6 5.10795 0.18114 ADHEr, ASPT,ATPS4r, LDH_D, MDH, PGL and/or G6PDHy 173 6 5.07418 0.20379 ADHEr,ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PPS 174 6 5.05205 0.12049 ADHEr,EDA and/or PGDHY, LDH_D, NADH6, PGI, PPCK 175 6 5.02318 0.17043 ACKrand/or PTAr, ADHEr, FUM, LDH_D, PFLi, PPCK 176 6 5.02318 0.17043 ACKrand/or PTAr, ADHEr, LDH_D, MDH, PFLi, PPCK 177 6 4.96023 0.0152 ADHEr,FRD and/or SUCD4, LDH_D, MDH, PYK, THD2 and/or GLUDy 178 6 4.960190.14703 ADHEr, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy 179 6 4.952270.18511 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, LDH_D, NADH6, PGI 1806 4.94767 0.18905 ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6, PFLi 181 64.94078 0.08876 ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK 182 6 4.883780.08315 ADHEr, ASPT, ATPS4r, FUM, LDH_D, THD2 and/or GLUDy 183 6 4.881380.0499 ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy184 6 4.8705 0.04951 ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, PPCK, THD2and/or GLUDy 185 6 4.86653 0.05103 ADHEr, FRD and/or SUCD4, LDH_D, MALS,MDH, THD2 and/or GLUDy 186 6 4.86653 0.05103 ADHEr, FRD and/or SUCD4,ICL, LDH_D, MDH, THD2 and/or GLUDy 187 6 4.85555 0.05127 ADHEr, ASPT,LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 188 6 4.81179 0.07918 ADHEr, FRDand/or SUCD4, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy 189 6 4.808340.05481 ADHEr, ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy 190 6 4.808340.05481 ADHEr, ASPT, LDH_D, MALS, MDH, THD2 and/or GLUDy 191 6 4.805460.08184 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy192 6 4.79254 0.05156 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D,MDH, THD2 and/or GLUDy 193 6 4.7879 0.08109 ADHEr, FRD and/or SUCD4,LDH_D, MALS, PPCK, THD2 and/or GLUDy 194 6 4.7879 0.08109 ADHEr, FRDand/or SUCD4, ICL, LDH_D, PPCK, THD2 and/or GLUDy 195 6 4.7863 0.15759ADHEr, LDH_D, NADH12, NADH6, PFLi, PGI 196 6 4.78022 0.08403 ADHEr, FRDand/or SUCD4, HEX1, LDH_D, MALS, THD2 and/or GLUDy 197 6 4.78022 0.08403ADHEr, FRD and/or SUCD4, HEX1, ICL, LDH_D, THD2 and/or GLUDy 198 64.75362 0.1717 ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS 199 6 4.744820.14204 ADHEr, FUM, LDH_D, PFLi, PGI, PPCK 200 6 4.74482 0.14204 ADHEr,LDH_D, MDH, PFLi, PGI, PPCK 201 6 4.71665 0.15772 ADHEr, FUM, LDH_D,NADH6, PFLi, PGI 202 6 4.71665 0.15772 ADHEr, LDH_D, MDH, NADH6, PFLi,PGI 203 6 4.67938 0.12258 ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH,THD2 and/or GLUDy 204 6 4.67056 0.08187 ACKr and/or PTAr, ADHEr, FRDand/or SUCD4, LDH_D, PPCK, THD2 and/or GLUDy 205 6 4.65509 0.08572 ACKrand/or PTAr, ADHEr, FRD and/or SUCD4, HEX1, LDH_D, THD2 and/or GLUDy 2066 4.65177 0.09959 ADHEr, ASPT, FUM, LDH_D, PPCK, PYK 207 6 4.648560.19942 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi 208 6 4.64527 0.14923ADHEr, LDH_D, MDH, NADH6, SUCOAS, THD2 and/or GLUDy 209 6 4.625010.24401 ADHEr, HEX1, LDH_D, NADH6, PFLi, THD2 and/or GLUDy 210 6 4.615690.16588 ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, RPE 211 6 4.583640.20034 ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE, SUCOAS 212 6 4.541490.17531 ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH, PYK 213 6 4.532770.1423 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, PPCK, THD2 and/or GLUDy214 6 4.53052 0.2259 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, RPE, SUCOAS215 6 4.52789 0.16756 ADHEr, ASPT, GLCpts, LDH_D, MDH, SUCOAS 216 64.52733 0.16694 ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH 217 6 4.505640.17546 ADHEr, ASPT, LDH_D, MDH, PYK, SUCOAS 218 6 4.48785 0.20333ADHEr, ASPT, LDH_D, MDH, RPE, SUCOAS 219 6 4.45673 0.0937 ADHEr, ATPS4r,EDA and/or PGDHY, LDH_D, PGI, PPCK 220 6 4.44964 0.1574 ADHEr, ASPT,FUM, LDH_D, MDH, THD2 and/or GLUDy 221 6 4.38551 0.17574 ADHEr, ASPT,GLCpts, LDH_D, MALS, MDH 222 6 4.38551 0.17574 ADHEr, ASPT, FUM, GLCpts,LDH_D, MDH 223 6 4.38551 0.17574 ADHEr, ASPT, GLCpts, ICL, LDH_D, MDH224 6 4.3659 0.18135 ADHEr, ASPT, LDH_D, MALS, MDH, PYK 225 6 4.36590.18135 ADHEr, ASPT, FUM, LDH_D, MDH, PYK 226 6 4.3659 0.18135 ADHEr,ASPT, ICL, LDH_D, MDH, PYK 227 6 4.35016 0.23064 ADHEr, FUM, HEX1,LDH_D, NADH6, SUCOAS 228 6 4.34833 0.1713 ADHEr, ATPS4r, LDH_D, NADH6,PFLi, PPCK 229 6 4.34286 0.23323 ADHEr, ASPT, FUM, HEX1, LDH_D, SUCOAS230 6 4.33401 0.17749 ACKr and/or PTAr, ADHEr, ASPT, GLCpts, LDH_D, MDH231 6 4.31842 0.21314 ADHEr, ASPT, ICL, LDH_D, MDH, RPE 232 6 4.318420.21314 ADHEr, ASPT, LDH_D, MALS, MDH, RPE 233 6 4.31842 0.21314 ADHEr,ASPT, FUM, LDH_D, MDH, RPE 234 6 4.31517 0.18251 ACKr and/or PTAr,ADHEr, ASPT, LDH_D, MDH, PYK 235 6 4.27405 0.08431 ADHEr, FRD and/orSUCD4, LDH_D, PPCK, RPE, THD2 and/or GLUDy 236 6 4.25521 0.17069 ACKrand/or PTAr, ADHEr, LDH_D, MDH, PPCK, PYK 237 6 4.25521 0.17069 ACKrand/or PTAr, ADHEr, FUM, LDH_D, PPCK, PYK 238 6 4.23738 0.22036 ADHEr,FUM, HEX1, LDH_D, PGI, THD5 239 6 4.23738 0.22036 ADHEr, HEX1, LDH_D,MDH, PGI, THD5 240 6 4.21937 0.24412 ADHEr, ASPT, FUM, HEX1, LDH_D, RPE241 6 4.20848 0.22638 ADHEr, ASPT, FUM, HEX1, LDH_D, THD2 and/or GLUDy242 6 4.1949 0.21515 ACKr and/or PTAr, ADHEr, FDH2, LDH_D, MDH, NADH6243 6 4.193 0.22615 ADHEr, ASPT, ATPS4r, FUM, HEX1, LDH_D 244 6 4.184640.18833 ADHEr, HEX1, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy 245 6 4.170220.12602 ADHEr, GLCpts, LDH_D, PFLi, PGI, PPCK 246 6 4.15452 0.23423 ACKrand/or PTAr, ACS, ADHEr, EDA and/or PGDHY, LDH_D, PGI 247 6 4.139580.1401 ADHEr, GLCpts, LDH_D, NADH6, PFLi, PGI 248 6 4.12806 0.09037ADHEr, ASPT, FUM, LDH_D, PPCK, THD2 and/or GLUDy 249 6 4.10281 0.14798ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK 250 6 4.09944 0.14944 ADHEr,LDH_D, NADH6, PFLi, PPCK, PYK 251 6 4.09149 0.20644 ADHEr, ASPT, ATPS4r,LDH_D, MDH, NADH6 252 6 4.08305 0.24974 ACKr and/or PTAr, ADHEr, FUM,HEX1, LDH_D, NADH6 253 6 4.07707 0.21283 ADHEr, ATPS4r, GLCpts, LDH_D,MDH, NADH6 254 6 4.07083 0.2483 ACKr and/or PTAr, ADHEr, FDH2, HEX1,LDH_D, NADH6 255 6 4.0547 0.15755 ADHEr, LDH_D, NADH6, PFLi, PGDH, PGI256 6 4.04976 0.14332 ADHEr, LDH_D, PFLi, PGDH, PGI, PPCK 257 6 4.049250.17118 ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK 258 6 4.04925 0.17118ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK 259 6 4.04921 0.20443 ADHEr,ATPS4r, FUM, LDH_D, PFLi, PPCK 260 6 4.04921 0.20443 ADHEr, ATPS4r,LDH_D, MDH, PFLi, PPCK 261 6 4.04701 0.15816 ADHEr, LDH_D, NADH6, PFLi,PGI, TAL 262 6 4.04276 0.14386 ADHEr, LDH_D, PFLi, PGI, PPCK, TAL 263 64.03993 0.15872 ADHEr, LDH_D, NADH6, PFLi, PGI, RPE 264 6 4.036320.14436 ADHEr, LDH_D, PFLi, PGI, PPCK, RPE 265 6 4.01325 0.18677 ADHEr,GLCpts, LDH_D, MDH, NADH6, PFLi 266 6 3.99577 0.19434 ADHEr, LDH_D, MDH,NADH12, NADH6, PFLi 267 6 3.97712 0.21218 ADHEr, ATPS4r, LDH_D, NADH6,PPCK, RPE 268 6 3.96781 0.21154 ADHEr, ATPS4r, LDH_D, NADH6, PPCK, TAL269 6 3.95767 0.21085 ADHEr, ATPS4r, LDH_D, NADH6, PGL and/or G6PDHy,PPCK 270 6 3.95767 0.21085 ADHEr, ATPS4r, LDH_D, NADH6, PGDH, PPCK 271 63.95117 0.22396 ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE 272 6 3.941430.22325 ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL 273 6 3.93082 0.22248ADHEr, ATPS4r, LDH_D, MDH, NADH6, PGDH 274 6 3.92007 0.30764 ADHEr,HEX1, LDH_D, PFLi, PPS, THD2 and/or GLUDy 275 6 3.90761 0.02965 ADHEr,EDA and/or PGDHY, LDH_D, MDH, PGI, THD2 and/or GLUDy 276 6 3.907610.02965 ADHEr, EDA and/or PGDHY, FUM, LDH_D, PGI, THD2 and/or GLUDy 2776 3.88276 0.27815 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, PFLi 278 6 3.876220.18142 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, PGI 279 6 3.85591 0.25493ADHEr, HEX1, LDH_D, NADH12, NADH6, PFLi 280 6 3.85214 0.25656 ADHEr,FUM, HEX1, LDH_D, NADH6, PFLi 281 6 3.80756 0.28917 ADHEr, LDH_D,NADH12, NADH6, PFLi, RPE 282 6 3.80403 0.29078 ADHEr, GLCpts, LDH_D,NADH6, PFLi, RPE 283 6 3.80376 0.2909 ADHEr, FUM, LDH_D, NADH6, PFLi,RPE 284 6 3.79095 0.29004 ADHEr, LDH_D, NADH12, NADH6, PFLi, TAL 285 63.78678 0.29189 ADHEr, FUM, LDH_D, NADH6, PFLi, TAL 286 6 3.745040.18693 ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PPCK 287 6 3.66618 0.23348ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6 288 6 3.65047 0.29574 ADHEr,ATPS4r, FDH2, LDH_D, NADH12, NADH6 289 6 3.64636 0.29624 ADHEr, ATPS4r,FDH2, GLCpts, LDH_D, NADH6 290 6 3.61821 0.2978 ADHEr, ATPS4r, FDH2,FUM, LDH_D, NADH6 291 6 3.59369 0.11801 ADHEr, EDA and/or PGDHY, GLUDy,LDH_D, MDH, THD2 and/or GLUDy 292 6 3.56749 0.21671 ADHEr, HEX1, LDH_D,PFLi, PPCK, THD2 and/or GLUDy 293 6 3.56467 0.20357 ADHEr, ATPS4r,LDH_D, MDH, NADH6, PPCK 294 6 3.56467 0.20357 ADHEr, ATPS4r, FUM, LDH_D,NADH6, PPCK 295 6 3.54915 0.24121 ADHEr, ATPS4r, EDA and/or PGDHY,LDH_D, MDH, THD2 and/or GLUDy 296 6 3.54182 0.17698 ADHEr, ASPT, EDAand/or PGDHY, LDH_D, PGI, THD5 297 6 3.52871 0.18935 ADHEr, ATPS4r,GLCpts, LDH_D, MDH, PPCK 298 6 3.52871 0.18935 ADHEr, ATPS4r, FUM,GLCpts, LDH_D, PPCK 299 6 3.48585 0.20551 ADHEr, ATPS4r, FDH2, LDH_D,NADH6, PPCK 300 6 3.46875 0.14666 ACKr and/or PTAr, ADHEr, LDH_D, PFLi,PGI, PPCK 301 6 3.39774 0.24244 ADHEr, ATPS4r, GLCpts, LDH_D, MDH, THD2and/or GLUDy 302 6 3.34501 0.09944 ACKr and/or PTAr, ADHEr, ATPS4r, FRDand/or SUCD4, LDH_D, PGI 303 6 3.21571 0.21219 ADHEr, LDH_D, MDH,NADH12, NADH6, SUCOAS 304 6 3.13882 0.32432 ADHEr, ATPS4r, HEX1, LDH_D,NADH6, PGL and/or G6PDHy 305 6 3.13882 0.32432 ADHEr, ATPS4r, HEX1,LDH_D, NADH6, PGDH 306 6 3.10937 0.32483 ADHEr, ATPS4r, HEX1, LDH_D,NADH6, TAL 307 6 3.08568 0.30099 ADHEr, ATPS4r, FUM, HEX1, LDH_D, NADH6308 6 3.08242 0.32529 ADHEr, ATPS4r, HEX1, LDH_D, NADH6, RPE 309 63.05396 0.31324 ADHEr, FUM, HEX1, LDH_D, PPS, THD2 and/or GLUDy 310 63.04084 0.1442 ADHEr, ATPS4r, GLCpts, LDH_D, PGI, PPCK 311 6 3.03310.26033 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, PGDH 312 6 2.955720.06686 ADHEr, ASPT, LDH_D, MDH, PGI, THD2 and/or GLUDy 313 6 2.952090.20166 ADHEr, ASPT, LDH_D, MDH, NADH6, PYK 314 6 2.93749 0.26369 ACKrand/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, TAL 315 6 2.93137 0.23554 ADHEr,EDA and/or PGDHY, FUM, LDH_D, PGI, THD5 316 6 2.93119 0.19434 ADHEr,ATPS4r, FUM, LDH_D, NADH6, THD2 and/or GLUDy 317 6 2.92428 0.21637ADHEr, ASPT, GLCpts, LDH_D, MDH, NADH6 318 6 2.90707 0.25203 ADHEr,ASPT, LDH_D, MDH, NADH6, RPE 319 6 2.88032 0.10845 ADHEr, ASPT, LDH_D,MDH, PYK, THD2 and/or GLUDy 320 6 2.85872 0.13782 ADHEr, ASPT, GLCpts,LDH_D, MDH, PGI 321 6 2.83622 0.13278 ADHEr, ASPT, GLCpts, LDH_D, MDH,THD2 and/or GLUDy 322 6 2.83037 0.25344 ACKr and/or PTAr, ADHEr, ATPS4r,HEX1, LDH_D, THD2 and/or GLUDy 323 6 2.73678 0.18763 ADHEr, ASPT, LDH_D,MDH, PPCK, PYK 324 6 2.73413 0.26033 ACKr and/or PTAr, ADHEr, ATPS4r,LDH_D, MDH, PGL and/or G6PDHy 325 6 2.70842 0.20327 ADHEr, ASPT, GLCpts,LDH_D, MDH, PPCK 326 6 2.68773 0.20429 ADHEr, FUM, LDH_D, NADH6, PGI,THD5 327 6 2.68773 0.20429 ADHEr, LDH_D, MDH, NADH6, PGI, THD5 328 62.6871 0.26683 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, RPE 329 62.64071 0.07956 ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, THD2 and/orGLUDy 330 6 2.61399 0.16269 ADHEr, LDH_D, MDH, NADH12, NADH6, THD2and/or GLUDy 331 6 2.60256 0.17034 ADHEr, LDH_D, NADH12, NADH6, PPCK,THD2 and/or GLUDy 332 6 2.56541 0.1952 ADHEr, FUM, GLCpts, LDH_D, NADH6,THD2 and/or GLUDy 333 6 2.53808 0.13335 ADHEr, ATPS4r, GLUDy, LDH_D,MDH, THD2 and/or GLUDy 334 6 2.53559 0.33664 ADHEr, ATPS4r, HEX1, LDH_D,PPS, THD2 and/or GLUDy 335 6 2.53316 0.24472 ADHEr, LDH_D, MDH, NADH6,PPCK, RPE 336 6 2.53316 0.24472 ADHEr, FUM, LDH_D, NADH6, PPCK, RPE 3376 2.53266 0.21711 ADHEr, FUM, LDH_D, NADH12, NADH6, THD2 and/or GLUDy338 6 2.50897 0.31991 ADHEr, HEX1, LDH_D, NADH6, PPS, THD2 and/or GLUDy339 6 2.47564 0.27754 ADHEr, ATPS4r, LDH_D, MDH, PGDH, TAL 340 6 2.374240.3231 ADHEr, GLU5K, HEX1, LDH_D, NADH6, THD2 and/or GLUDy 341 6 2.374240.3231 ADHEr, G5SD, HEX1, LDH_D, NADH6, THD2 and/or GLUDy 342 6 2.374180.32314 ADHEr, ASNS2, HEX1, LDH_D, NADH6, THD2 and/or GLUDy 343 62.35165 0.11344 ADHEr, FUM, LDH_D, PPCK, PYK, THD2 and/or GLUDy 344 62.35165 0.11344 ADHEr, LDH_D, MDH, PPCK, PYK, THD2 and/or GLUDy 345 62.28934 0.18186 ADHEr, HEX1, LDH_D, PPS, RPE, THD2 and/or GLUDy 346 62.28203 0.17727 ADHEr, HEX1, LDH_D, PPS, TAL, THD2 and/or GLUDy 347 62.27443 0.17249 ADHEr, HEX1, LDH_D, PGDH, PPS, THD2 and/or GLUDy 348 62.27443 0.17249 ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPS, THD2 and/orGLUDy 349 6 2.23539 0.22843 ADHEr, HEX1, LDH_D, PPCK, RPE, THD2 and/orGLUDy 350 6 2.2276 0.23515 ADHEr, FUM, HEX1, LDH_D, RPE, THD2 and/orGLUDy 351 6 2.22058 0.22724 ADHEr, HEX1, LDH_D, PPCK, TAL, THD2 and/orGLUDy 352 6 2.20452 0.22594 ADHEr, HEX1, LDH_D, PGDH, PPCK, THD2 and/orGLUDy 353 6 2.20452 0.22594 ADHEr, HEX1, LDH_D, PGL and/or G6PDHy, PPCK,THD2 and/or GLUDy 354 6 2.10135 0.3242 ADHEr, FUM, HEX1, LDH_D, TAL,THD2 and/or GLUDy 355 6 2.02123 0.34714 ADHEr, ATPS4r, HEX1, LDH_D, RPE,THD2 and/or GLUDy 356 6 1.98114 0.30095 ACKr and/or PTAr, ADHEr, ATPS4r,FUM, LDH_D, THD2 and/or GLUDy 357 6 1.96512 0.30664 ADHEr, ATPS4r, HEX1,LDH_D, MDH, PFLi 358 6 1.96163 0.34738 ADHEr, ATPS4r, HEX1, LDH_D, TAL,THD2 and/or GLUDy 359 6 1.94766 0.26881 ADHEr, ATPS4r, LDH_D, MDH, PGLand/or G6PDHy, PPS 360 6 1.84554 0.35787 ADHEr, HEX1, LDH_D, PFLi, PPS,RPE 361 6 1.84094 0.27637 ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, FUM,LDH_D 362 6 1.82413 0.31504 ADHEr, ATPS4r, HEX1, LDH_D, PFLi, RPE 363 61.82134 0.30945 ADHEr, HEX1, LDH_D, MDH, PFLi, RPE 364 6 1.79836 0.35901ADHEr, HEX1, LDH_D, PFLi, PPS, TAL 365 6 1.74423 0.2066 ADHEr, ASPT,ATPS4r, LDH_D, PGI, THD5 366 6 1.63108 0.27824 ACKr and/or PTAr, ADHEr,LDH_D, MDH, PGDH, SUCOAS 367 6 1.63108 0.27824 ACKr and/or PTAr, ADHEr,LDH_D, MDH, PGL and/or G6PDHy, SUCOAS 368 6 1.59283 0.279 ACKr and/orPTAr, ADHEr, LDH_D, MDH, SUCOAS, TAL 369 6 1.55776 0.2797 ACKr and/orPTAr, ADHEr, LDH_D, MDH, RPE, SUCOAS 370 6 1.35784 0.33828 ADHEr, FUM,LDH_D, PPS, SUCOAS, THD2 and/or GLUDy 371 6 1.3281 0.25919 ACKr and/orPTAr, ADHEr, ASPT, FUM, LDH_D, SUCOAS 372 6 1.25004 0.31142 ACKr and/orPTAr, ADHEr, FUM, LDH_D, SUCOAS, THD2 and/or GLUDy 373 6 1.16323 0.25946ADHEr, FRD and/or SUCD4, GLYCL, LDH_D, PGL and/or G6PDHy, TAL 374 61.16323 0.25946 ADHEr, FRD and/or SUCD4, GLYCL, LDH_D, PGDH, TAL 375 60.94735 0.32316 ACKr and/or PTAr, ADHEr, HEX1, LDH_D, PPS, RPE 376 60.88343 0.32336 ACKr and/or PTAr, ADHEr, HEX1, LDH_D, PPS, TAL 377 60.83542 0.27562 ADHEr, ASPT, FUM, LDH_D, PGDH, TAL 378 6 0.83542 0.27562ADHEr, ASPT, FUM, LDH_D, PGL and/or G6PDHy, TAL 379 6 0.81371 0.28445ACKr and/or PTAr, ADHEr, ASPT, FUM, ICL, LDH_D 380 6 0.81371 0.28445ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D, MALS 381 6 0.40293 0.37998ACKr and/or PTAr, ADHEr, CITL, ICL, LDH_D, SUCOAS 382 6 0.40293 0.37998ACKr and/or PTAr, ADHEr, CITL, LDH_D, MALS, SUCOAS 383 7 6.87929 0.03662ADHEr, ASPT, LDH_D, MDH, PFLi, PGL and/or G6PDHy, PYK 384 7 6.879290.03662 ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PFLi, PYK 385 76.82182 0.04856 ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PFLi, THD2and/or GLUDy 386 7 6.7498 0.06331 ADHEr, ASPT, LDH_D, MDH, PFLi, PPCK,THD2 and/or GLUDy 387 7 6.70416 0.07266 ADHEr, ASPT, LDH_D, MDH, NADH6,PFLi, THD2 and/or GLUDy 388 7 6.67113 0.07943 ADHEr, ASPT, GLCpts,LDH_D, MDH, PFLi, THD2 and/or GLUDy 389 7 6.65401 0.08294 ADHEr, ATPS4r,FDH2, LDH_D, MDH, NADH6, PGL and/or G6PDHy 390 7 6.45147 0.12443 ADHEr,ATPS4r, LDH_D, MDH, NADH6, PFLi, PGL and/or G6PDHy 391 7 6.44861 0.08805ADHEr, ASPT, ATPS4r, LDH_D, MALS, MDH, PGL and/or G6PDHy 392 7 6.448610.08805 ADHEr, ASPT, ATPS4r, ICL, LDH_D, MDH, PGL and/or G6PDHy 393 76.44861 0.08805 ADHEr, ASPT, ATPS4r, FUM, LDH_D, MDH, PGL and/or G6PDHy394 7 6.15823 0.07915 ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MALS, MDH 3957 6.15823 0.07915 ADHEr, ASPT, ATPS4r, GLCpts, ICL, LDH_D, MDH 396 76.15823 0.07915 ADHEr, ASPT, ATPS4r, FUM, GLCpts, LDH_D, MDH 397 76.14605 0.07491 ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, SUCOAS 398 76.1414 0.1578 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, NADH6, PGDH399 7 6.09037 0.16006 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH,NADH6, TAL 400 7 6.08683 0.10358 ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi,THD2 and/or GLUDy 401 7 6.0464 0.06183 ADHEr, ASPT, GLCpts, LDH_D, MDH,PFLi, PGI 402 7 6.04253 0.16218 ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D,MDH, NADH6, RPE 403 7 5.86291 0.1062 ADHEr, ASPT, EDA and/or PGDHY, ICL,LDH_D, MDH, THD2 and/or GLUDy 404 7 5.86291 0.1062 ADHEr, ASPT, EDAand/or PGDHY, LDH_D, MALS, MDH, THD2 and/or GLUDy 405 7 5.79637 0.08575ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, NADH6, PGI 406 7 5.746420.10456 ADHEr, EDA and/or PGDHY, FRD and/or SUCD4, GLCpts, LDH_D, MDH,THD2 and/or GLUDy 407 7 5.71711 0.12348 ADHEr, ASPT, ATPS4r, LDH_D, MDH,NADH6, PFLi 408 7 5.71588 0.18172 ADHEr, ATPS4r, FRD and/or SUCD4, HEX1,LDH_D, PGL and/or G6PDHy, PPS 409 7 5.71588 0.18172 ADHEr, ATPS4r, FRDand/or SUCD4, HEX1, LDH_D, PGDH, PPS 410 7 5.70575 0.12457 ADHEr, ASPT,ATPS4r, GLCpts, LDH_D, MDH, PFLi 411 7 5.69395 0.07981 ADHEr, ATPS4r,EDA and/or PGDHY, FRD and/or SUCD4, LDH_D, MDH, PPCK 412 7 5.688260.10159 ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, PFLi, PGI, PPCK 413 75.6857 0.18276 ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PPS, TAL414 7 5.67373 0.1078 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, LDH_D,PFLi, PGI, PPCK 415 7 5.67231 0.09516 ACKr and/or PTAr, ADHEr, ATPS4r,EDA and/or PGDHY, FRD and/or SUCD4, LDH_D, MDH 416 7 5.65788 0.18372ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, PPS, RPE 417 7 5.651080.11748 ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, NADH6, PFLi, PGI 418 75.64055 0.12198 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, LDH_D, NADH6,PFLi, PGI 419 7 5.63038 0.12633 ADHEr, LDH_D, MDH, NADH12, NADH6, PFLi,PGI 420 7 5.63037 0.14151 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, PPS,SUCOAS, THD2 and/or GLUDy 421 7 5.61946 0.14377 ADHEr, ATPS4r, EDAand/or PGDHY, FRD and/or SUCD4, LDH_D, MDH, SUCOAS 422 7 5.61946 0.14377ADHEr, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, NADH6, SUCOAS 423 7 5.607130.15264 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MALS, PPS, THD2 and/orGLUDy 424 7 5.60713 0.15264 ADHEr, FRD and/or SUCD4, HEX1, ICL, LDH_D,PPS, THD2 and/or GLUDy 425 7 5.60017 0.14365 ADHEr, ATPS4r, FRD and/orSUCD4, LDH_D, MDH, PGDH, SUCOAS 426 7 5.59518 0.14138 ADHEr, FUM, LDH_D,NADH12, NADH6, PFLi, PGI 427 7 5.59117 0.14212 ADHEr, ATPS4r, FRD and/orSUCD4, FUM, LDH_D, MDH, PGDH 428 7 5.58992 0.13977 ADHEr, ATPS4r, FRDand/or SUCD4, LDH_D, MDH, PGDH, TAL 429 7 5.57521 0.1433 ADHEr, HEX1,LDH_D, MDH, PPS, SUCOAS, THD2 and/or GLUDy 430 7 5.57521 0.1433 ADHEr,FUM, HEX1, LDH_D, PPS, SUCOAS, THD2 and/or GLUDy 431 7 5.56382 0.059ADHEr, HEX1, LDH_D, PFLi, PGI, PPS, THD2 and/or GLUDy 432 7 5.557140.14423 ADHEr, ASPT, EDA and/or PGDHY, FUM, LDH_D, MDH, THD2 and/orGLUDy 433 7 5.5551 0.14719 ADHEr, HEX1, LDH_D, MDH, NADH6, SUCOAS, THD2and/or GLUDy 434 7 5.54832 0.1485 ADHEr, ASPT, HEX1, LDH_D, MDH, SUCOAS,THD2 and/or GLUDy 435 7 5.54749 0.08705 ADHEr, LDH_D, NADH12, NADH6,PFLi, PPCK, THD2 and/or GLUDy 436 7 5.54 0.14449 ADHEr, ATPS4r, LDH_D,MDH, NADH6, PGDH, SUCOAS 437 7 5.53962 0.15019 ADHEr, ASPT, ATPS4r, EDAand/or PGDHY, LDH_D, MDH, SUCOAS 438 7 5.53831 0.08799 ADHEr, LDH_D,MDH, NADH6, PFLi, PPCK, THD2 and/or GLUDy 439 7 5.53831 0.08799 ADHEr,FUM, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy 440 7 5.53405 0.05732ACKr and/or PTAr, ADHEr, ASPT, EDA and/or PGDHY, LDH_D, MDH, PGI 441 75.52463 0.06237 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH, PGI442 7 5.51713 0.16489 ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, SUCOAS 4437 5.51587 0.14532 ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS, TAL 444 75.51034 0.14586 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PFLi, PGDH 445 75.51034 0.14586 ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6, PGDH 446 75.5014 0.07482 ADHEr, ASPT, EDA and/or PGDHY, GLCpts, LDH_D, MDH, PGI447 7 5.49363 0.14609 ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE, SUCOAS 4487 5.49315 0.18499 ACKr and/or PTAr, ADHEr, EDA and/or PGDHY, HEX1,LDH_D, PFLi, PGI 449 7 5.48581 0.14671 ADHEr, ATPS4r, LDH_D, MDH, NADH6,PFLi, TAL 450 7 5.48581 0.14671 ADHEr, ATPS4r, FDH2, LDH_D, MDH, NADH6,TAL 451 7 5.47686 0.19195 ADHEr, EDA and/or PGDHY, LDH_D, MDH, NADH6,PGI, THD5 452 7 5.47123 0.12153 ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK,THD2 and/or GLUDy 453 7 5.47123 0.12153 ADHEr, FUM, GLCpts, LDH_D, PFLi,PPCK, THD2 and/or GLUDy 454 7 5.46318 0.14749 ADHEr, ATPS4r, FDH2,LDH_D, MDH, NADH6, RPE 455 7 5.46318 0.14749 ADHEr, ATPS4r, LDH_D, MDH,NADH6, PFLi, RPE 456 7 5.43967 0.13732 ADHEr, GLCpts, LDH_D, MDH, NADH6,PFLi, THD2 and/or GLUDy 457 7 5.41442 0.15603 ADHEr, ASPT, FUM, HEX1,LDH_D, MDH, THD2 and/or GLUDy 458 7 5.41442 0.15603 ADHEr, ASPT, HEX1,LDH_D, MALS, MDH, THD2 and/or GLUDy 459 7 5.41442 0.15603 ADHEr, ASPT,HEX1, ICL, LDH_D, MDH, THD2 and/or GLUDy 460 7 5.32088 0.07204 ADHEr,FRD and/or SUCD4, HEX1, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy 461 75.29376 0.07385 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, MALS, PPCK, THD2and/or GLUDy 462 7 5.29376 0.07385 ADHEr, FRD and/or SUCD4, HEX1, ICL,LDH_D, PPCK, THD2 and/or GLUDy 463 7 5.29342 0.0731 ADHEr, HEX1, LDH_D,MDH, PPCK, SUCOAS, THD2 and/or GLUDy 464 7 5.29342 0.0731 ADHEr, FUM,HEX1, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy 465 7 5.20741 0.07603ADHEr, ASPT, ATPS4r, FUM, LDH_D, PGDH, PPCK 466 7 5.20741 0.07603 ADHEr,ASPT, ATPS4r, FUM, LDH_D, PGL and/or G6PDHy, PPCK 467 7 5.20289 0.16585ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PFLi, RPE 468 7 5.194690.07664 ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK, TAL 469 7 5.18818 0.16709ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH, PFLi, TAL 470 7 5.182920.0772 ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK, RPE 471 7 5.18092 0.10975ADHEr, FUM, GLCpts, LDH_D, PFLi, PGI, PPCK 472 7 5.18092 0.10975 ADHEr,GLCpts, LDH_D, MDH, PFLi, PGI, PPCK 473 7 5.18025 0.13169 ACKr and/orPTAr, ADHEr, GLCpts, LDH_D, MDH, PFLi, PPCK 474 7 5.18025 0.13169 ACKrand/or PTAr, ADHEr, FUM, GLCpts, LDH_D, PFLi, PPCK 475 7 5.17526 0.14527ADHEr, ATPS4r, LDH_D, MDH, NADH12, NADH6, PFLi 476 7 5.15999 0.12483ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi, PGI 477 7 5.15742 0.1471 ADHEr,ATPS4r, GLCpts, LDH_D, MDH, NADH6, PFLi 478 7 5.13283 0.14064 ACKrand/or PTAr, ADHEr, LDH_D, MDH, PFLi, PGI, PPCK 479 7 5.13283 0.14064ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFLi, PGI, PPCK 480 7 5.111280.1487 ACKr and/or PTAr, ADHEr, GLCpts, LDH_D, MDH, NADH6, PFLi 481 75.10917 0.0835 ADHEr, EDA and/or PGDHY, GLCpts, LDH_D, NADH6, PGI, PPCK482 7 5.10795 0.18114 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D, MDH,PGDH 483 7 5.08422 0.18199 ADHEr, ASPT, ATPS4r, EDA and/or PGDHY, LDH_D,MDH, TAL 484 7 5.08162 0.15602 ACKr and/or PTAr, ADHEr, LDH_D, NADH12,NADH6, PFLi, PGI 485 7 5.08154 0.15604 ACKr and/or PTAr, ADHEr, LDH_D,MDH, NADH6, PFLi, PGI 486 7 5.07957 0.15652 ACKr and/or PTAr, ADHEr,FUM, LDH_D, NADH6, PFLi, PGI 487 7 5.06238 0.18278 ADHEr, ASPT, ATPS4r,EDA and/or PGDHY, LDH_D, MDH, RPE 488 7 5.04933 0.08188 ADHEr, ATPS4r,FDH2, LDH_D, NADH6, PPCK, SUCOAS 489 7 5.04086 0.10883 ACKr and/or PTAr,ADHEr, LDH_D, MDH, NADH6, PGDH, PGI 490 7 5.03782 0.10979 ACKr and/orPTAr, ADHEr, LDH_D, MDH, NADH6, PGI, TAL 491 7 5.03499 0.11069 ACKrand/or PTAr, ADHEr, LDH_D, MDH, NADH6, PGI, RPE 492 7 5.0347 0.123 ACKrand/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS 493 7 5.023250.08242 ADHEr, ATPS4r, LDH_D, MDH, NADH6, PPCK, SUCOAS 494 7 5.023250.08242 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PPCK, SUCOAS 495 7 5.022330.1115 ADHEr, GLCpts, LDH_D, NADH6, PFLi, PPCK, THD2 and/or GLUDy 496 75.00367 0.01301 ADHEr, ATPS4r, LDH_D, MDH, NADH6, SUCOAS, THD2 and/orGLUDy 497 7 5.00099 0.08371 ADHEr, ASPT, ATPS4r, FUM, LDH_D, PPCK,SUCOAS 498 7 4.9785 0.07565 ADHEr, ATPS4r, FDH2, LDH_D, NADH6, SUCOAS,THD2 and/or GLUDy 499 7 4.97218 0.04755 ADHEr, EDA and/or PGDHY, HEX1,LDH_D, NADH6, PGI, THD2 and/or GLUDy 500 7 4.96921 0.01295 ADHEr, FRDand/or SUCD4, GLUDy, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 501 7 4.967160.01165 ADHEr, GLUDy, HEX1, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 502 74.96611 0.01426 ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, SUCOAS,THD2 and/or GLUDy 503 7 4.96543 0.01322 ADHEr, FRD and/or SUCD4, GLUDy,ICL, LDH_D, MDH, THD2 and/or GLUDy 504 7 4.96543 0.01322 ADHEr, FRDand/or SUCD4, GLUDy, LDH_D, MALS, MDH, THD2 and/or GLUDy 505 7 4.963350.01401 ADHEr, AKGD, FRD and/or SUCD4, ICL, LDH_D, PPCK, THD2 and/orGLUDy 506 7 4.96335 0.01401 ADHEr, AKGD, FRD and/or SUCD4, LDH_D, MALS,PPCK, THD2 and/or GLUDy 507 7 4.96309 0.0131 ADHEr, GLUDy, LDH_D, MDH,NADH6, SUCOAS, THD2 and/or GLUDy 508 7 4.96271 0.01323 ADHEr, ASPT,GLUDy, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 509 7 4.96187 0.01458ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MALS, MDH, THD2 and/or GLUDy 5107 4.96187 0.01458 ADHEr, FRD and/or SUCD4, GLCpts, ICL, LDH_D, MDH, THD2and/or GLUDy 511 7 4.95873 0.01465 ADHEr, ASPT, GLCpts, LDH_D, MDH,SUCOAS, THD2 and/or GLUDy 512 7 4.95686 0.01531 ADHEr, ASPT, LDH_D, MDH,PYK, SUCOAS, THD2 and/or GLUDy 513 7 4.95085 0.01406 ADHEr, ASPT, GLUDy,LDH_D, MALS, MDH, THD2 and/or GLUDy 514 7 4.95085 0.01406 ADHEr, ASPT,GLUDy, ICL, LDH_D, MDH, THD2 and/or GLUDy 515 7 4.94529 0.0136 ACKrand/or PTAr, ADHEr, FRD and/or SUCD4, GLUDy, LDH_D, MDH, THD2 and/orGLUDy 516 7 4.94524 0.01566 ADHEr, ASPT, GLCpts, LDH_D, MALS, MDH, THD2and/or GLUDy 517 7 4.94524 0.01566 ADHEr, ASPT, GLCpts, ICL, LDH_D, MDH,THD2 and/or GLUDy 518 7 4.94393 0.07737 ADHEr, ATPS4r, FUM, LDH_D,NADH6, SUCOAS, THD2 and/or GLUDy 519 7 4.94353 0.01615 ADHEr, ASPT, FUM,LDH_D, MDH, PYK, THD2 and/or GLUDy 520 7 4.94353 0.01615 ADHEr, ASPT,ICL, LDH_D, MDH, PYK, THD2 and/or GLUDy 521 7 4.94353 0.01615 ADHEr,ASPT, LDH_D, MALS, MDH, PYK, THD2 and/or GLUDy 522 7 4.94073 0.01473ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, THD2and/or GLUDy 523 7 4.9406 0.01618 ACKr and/or PTAr, ADHEr, LDH_D, MDH,NADH6, PYK, THD2 and/or GLUDy 524 7 4.93746 0.13888 ADHEr, ASPT, ATPS4r,GLCpts, LDH_D, MDH, NADH6 525 7 4.93038 0.19331 ACKr and/or PTAr, ADHEr,ATPS4r, LDH_D, MDH, NADH6, PGDH 526 7 4.92604 0.19438 ACKr and/or PTAr,ADHEr, ATPS4r, LDH_D, MDH, NADH6, TAL 527 7 4.92485 0.04913 ACKr and/orPTAr, ADHEr, HEX1, LDH_D, NADH6, PGI, THD2 and/or GLUDy 528 7 4.922030.19537 ACKr and/or PTAr, ADHEr, ATPS4r, LDH_D, MDH, NADH6, RPE 529 74.89572 0.18301 ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH, PGDH530 7 4.88586 0.18375 ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D, MDH,TAL 531 7 4.8768 0.18443 ACKr and/or PTAr, ADHEr, ASPT, ATPS4r, LDH_D,MDH, RPE 532 7 4.87463 0.19612 ACKr and/or PTAr, ADHEr, LDH_D, MDH,NADH6, PGI, THD5 533 7 4.87463 0.19612 ACKr and/or PTAr, ADHEr, FUM,LDH_D, NADH6, PGI, THD5 534 7 4.85806 0.05038 ADHEr, LDH_D, MDH, NADH12,NADH6, SUCOAS, THD2 and/or GLUDy 535 7 4.85603 0.21579 ADHEr, ASPT,ATPS4r, FUM, HEX1, LDH_D, PPS 536 7 4.84975 0.05712 ADHEr, FRD and/orSUCD4, LDH_D, MDH, PGL and/or G6PDHy, PPCK, PYK 537 7 4.84975 0.05712ADHEr, EDA and/or PGDHY, FRD and/or SUCD4, LDH_D, MDH, PPCK, PYK 538 74.81569 0.05271 ADHEr, ASPT, FUM, GLCpts, LDH_D, PPCK, THD2 and/or GLUDy539 7 4.81323 0.1198 ADHEr, ATPS4r, GLCpts, LDH_D, NADH6, PFLi, PPCK 5407 4.81194 0.09049 ADHEr, FRD and/or SUCD4, LDH_D, PPCK, PYK, RPE, SUCOAS541 7 4.81179 0.07918 ADHEr, LDH_D, NADH12, NADH6, PPCK, SUCOAS, THD2and/or GLUDy 542 7 4.79812 0.05498 ACKr and/or PTAr, ADHEr, FUM, LDH_D,MDH, NADH6, THD2 and/or GLUDy 543 7 4.79812 0.05498 ACKr and/or PTAr,ADHEr, LDH_D, MALS, MDH, NADH6, THD2 and/or GLUDy 544 7 4.79812 0.05498ACKr and/or PTAr, ADHEr, ICL, LDH_D, MDH, NADH6, THD2 and/or GLUDy 545 74.79409 0.12192 ADHEr, ATPS4r, LDH_D, NADH12, NADH6, PFLi, PPCK 546 74.79101 0.05495 ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D, MDH, THD2and/or GLUDy 547 7 4.78296 0.12315 ADHEr, ATPS4r, LDH_D, MDH, NADH6,PFLi, PPCK 548 7 4.78296 0.12315 ADHEr, ATPS4r, FUM, LDH_D, NADH6, PFLi,PPCK 549 7 4.77588 0.08569 ADHEr, AKGD, FRD and/or SUCD4, LDH_D, MALS,MDH, THD2 and/or GLUDy 550 7 4.77588 0.08569 ADHEr, AKGD, FRD and/orSUCD4, ICL, LDH_D, MDH, THD2 and/or GLUDy 551 7 4.77561 0.07964 ADHEr,FUM, LDH_D, NADH6, PPCK, SUCOAS, THD2 and/or GLUDy 552 7 4.77561 0.07964ADHEr, LDH_D, MDH, NADH6, PPCK, SUCOAS, THD2 and/or GLUDy 553 7 4.773290.08047 ADHEr, ASPT, LDH_D, MDH, PPCK, SUCOAS, THD2 and/or GLUDy 554 74.77329 0.08047 ADHEr, ASPT, FUM, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy555 7 4.7705 0.16052 ADHEr, ATPS4r, FDH2, GLCpts, LDH_D, MDH, NADH6 5567 4.76518 0.08334 ADHEr, FUM, HEX1, LDH_D, NADH6, SUCOAS, THD2 and/orGLUDy 557 7 4.76374 0.0222 ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH, THD2and/or GLUDy 558 7 4.76264 0.08425 ADHEr, ASPT, FUM, HEX1, LDH_D,SUCOAS, THD2 and/or GLUDy 559 7 4.74028 0.09218 ADHEr, FUM, LDH_D,NADH6, PPCK, PYK, SUCOAS 560 7 4.74028 0.09218 ADHEr, LDH_D, MDH, NADH6,PPCK, PYK, SUCOAS 561 7 4.73717 0.09329 ADHEr, ASPT, LDH_D, MDH, PPCK,PYK, SUCOAS 562 7 4.73717 0.09329 ADHEr, ASPT, FUM, LDH_D, PPCK, PYK,SUCOAS 563 7 4.71257 0.12092 ADHEr, GLCpts, LDH_D, MDH, NADH6, SUCOAS,THD2 and/or GLUDy 564 7 4.69803 0.08636 ADHEr, ASPT, FUM, LDH_D, MALS,PPCK, THD2 and/or GLUDy 565 7 4.69803 0.08636 ADHEr, ASPT, FUM, ICL,LDH_D, PPCK, THD2 and/or GLUDy 566 7 4.69803 0.08636 ADHEr, ASPT, LDH_D,MALS, MDH, PPCK, THD2 and/or GLUDy 567 7 4.69803 0.08636 ADHEr, ASPT,ICL, LDH_D, MDH, PPCK, THD2 and/or GLUDy 568 7 4.68998 0.14917 ADHEr,FRD and/or SUCD4, LDH_D, MDH, RPE, SUCOAS, THD2 and/or GLUDy 569 74.68111 0.0912 ADHEr, ASPT, FUM, HEX1, LDH_D, MALS, THD2 and/or GLUDy570 7 4.68111 0.0912 ADHEr, ASPT, FUM, HEX1, ICL, LDH_D, THD2 and/orGLUDy 571 7 4.68063 0.05195 ACKr and/or PTAr, ADHEr, LDH_D, MDH, NADH6,SUCOAS, THD2 and/or GLUDy 572 7 4.67974 0.09159 ADHEr, AKGD, ASPT,LDH_D, MALS, MDH, THD2 and/or GLUDy 573 7 4.67974 0.09159 ADHEr, AKGD,ASPT, ICL, LDH_D, MDH, THD2 and/or GLUDy 574 7 4.6782 0.13538 ACKrand/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, THD2 and/orGLUDy 575 7 4.67079 0.08656 ACKr and/or PTAr, ADHEr, ASPT, FUM, LDH_D,PPCK, THD2 and/or GLUDy 576 7 4.66866 0.1492 ADHEr, FRD and/or SUCD4,LDH_D, MDH, SUCOAS, TAL, THD2 and/or GLUDy 577 7 4.66526 0.1329 ADHEr,HEX1, LDH_D, MDH, RPE, SUCOAS, THD2 and/or GLUDy 578 7 4.65177 0.09959ADHEr, ASPT, LDH_D, MALS, MDH, PPCK, PYK 579 7 4.65177 0.09959 ADHEr,ASPT, ICL, LDH_D, MDH, PPCK, PYK 580 7 4.65148 0.09163 ACKr and/or PTAr,ADHEr, ASPT, FUM, HEX1, LDH_D, THD2 and/or GLUDy 581 7 4.64795 0.13132ADHEr, ATPS4r, HEX1, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 582 7 4.647170.13258 ADHEr, HEX1, LDH_D, MDH, SUCOAS, TAL, THD2 and/or GLUDy 583 74.63555 0.16411 ADHEr, FRD and/or SUCD4, GLCpts, LDH_D, MDH, SUCOAS, TAL584 7 4.62743 0.13223 ADHEr, HEX1, LDH_D, MDH, PGDH, SUCOAS, THD2 and/orGLUDy 585 7 4.62722 0.10661 ADHEr, AKGD, ASPT, ICL, LDH_D, MDH, PYK 5867 4.62722 0.10661 ADHEr, AKGD, ASPT, LDH_D, MALS, MDH, PYK 587 7 4.614420.12597 ACKr and/or PTAr, ADHEr, ATPS4r, FUM, LDH_D, PPCK, THD2 and/orGLUDy 588 7 4.60692 0.10476 ACKr and/or PTAr, ADHEr, ASPT, ATPS4r,LDH_D, MDH, PGI 589 7 4.60239 0.15202 ADHEr, AKGD, FRD and/or SUCD4,HEX1, LDH_D, MALS, THD2 and/or GLUDy 590 7 4.60239 0.15202 ADHEr, AKGD,FRD and/or SUCD4, HEX1, ICL, LDH_D, THD2 and/or GLUDy 591 7 4.597770.03574 ACKr and/or PTAr, ADHEr, GLUDy, LDH_D, MDH, NADH6, THD2 and/orGLUDy 592 7 4.59693 0.17398 ADHEr, ASPT, FRD and/or SUCD4, LDH_D, MDH,PYK, RPE 593 7 4.59408 0.12607 ADHEr, ASPT, ATPS4r, GLCpts, LDH_D, MDH,PPCK 594 7 4.588 0.17333 ADHEr, ASPT, LDH_D, MDH, NADH6, PYK, SUCOAS 5957 4.58269 0.16568 ADHEr, ASPT, GLCpts, LDH_D, MDH, RPE, SUCOAS 596 74.5614 0.17413 ADHEr, ASPT, LDH_D, MDH, PYK, RPE, SUCOAS 597 7 4.555140.12723 ADHEr, ASPT, FUM, GLCpts, LDH_D, MDH, THD2 and/or GLUDy 598 74.54823 0.19006 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, HEX1, LDH_D,SUCOAS, THD2 and/or GLUDy 599 7 4.54674 0.19069 ACKr and/or PTAr, ADHEr,ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, SUCOAS 600 7 4.53866 0.20774ADHEr, FRD and/or SUCD4, HEX1, LDH_D, SUCOAS, TAL, THD2 and/or GLUDy 6017 4.53699 0.1728 ADHEr, FRD and/or SUCD4, FUM, LDH_D, MDH, PYK, TAL 6027 4.53586 0.209 ADHEr, ATPS4r, FRD and/or SUCD4, HEX1, LDH_D, SUCOAS,TAL 603 7 4.51849 0.13149 ADHEr, ATPS4r, FDH2, GLCpts, LDH_D, NADH6,PPCK 604 7 4.51845 0.20259 ADHEr, FDH2, LDH_D, MDH, NADH12, NADH6,SUCOAS 605 7 4.50909 0.20652 ADHEr, ATPS4r, FRD and/or SUCD4, HEX1,LDH_D, SUCOAS, THD2 and/or GLUDy 606 7 4.50549 0.20804 ADHEr, FRD and/orSUCD4, GLU5K, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy 607 7 4.505490.20804 ADHEr, FRD and/or SUCD4, G5SD, HEX1, LDH_D, SUCOAS, THD2 and/orGLUDy 608 7 4.50543 0.20807 ADHEr, ASNS2, FRD and/or SUCD4, HEX1, LDH_D,SUCOAS, THD2 and/or GLUDy 609 7 4.50378 0.20876 ADHEr, HEX1, LDH_D,NADH12, NADH6, SUCOAS, THD2 and/or GLUDy 610 7 4.50198 0.20952 ADHEr,ATPS4r, FDH2, HEX1, LDH_D, NADH6, SUCOAS 611 3 0.669528208 0.263200019ADHEr, FADH4, LDH_D 612 3 0.110808857 0.249569497 ADHEr, LDH_D, PFKand/or FBA and/or TPI 613 4 0.93088625 0.384018912 ADHEr, ATPS4r, CBMK2,LDH_D 614 4 1.163912961 0.378757536 ADHEr, ATPS4r, LDH_D, TKT2 615 44.552254757 0.171189177 ADHEr, FADH4, LDH_D, MDH 616 4 0.7109355680.257359864 ADHEr, FADH4, LDH_D, RPE 617 4 0.702400877 0.260125701ADHEr, FADH4, LDH_D, TAL 618 4 0.719290675 0.254652225 ADHEr, FADH4,LDH_D, TKT2 619 4 2.283994469 0.165173433 ADHEr, LDH_D, PFK and/or FBAand/or TPI, PPCK 620 4 0.18159615 0.246009686 ADHEr, LDH_D, PFK and/orFBA and/or TPI, TKT2 621 4 4.555303308 0.170023606 ADHEr, FRD and/orSUCD4, LDH_D, ME2 622 4 0.710935568 0.257359864 ADHEr, FRD and/or SUCD4,LDH_D, RPE 623 4 0.702400877 0.260125701 ADHEr, FRD and/or SUCD4, LDH_D,TAL 624 4 0.753832099 0.262897433 ADHEr, FRD and/or SUCD4, LDH_D, THD2and/or GLUDy 625 4 0.719290675 0.254652225 ADHEr, FRD and/or SUCD4,LDH_D, TKT2 626 4 2.165485624 0.255793845 ADHEr, FUM, LDH_D, PPCK 627 42.279039418 0.168962421 ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI628 4 0.24281895 0.340878264 ADHEr, HEX1, LDH_D, MDH 629 4 2.1654856240.255793845 ADHEr, LDH_D, MDH, PPCK 630 4 4.555303308 0.170023606 ADHEr,FADH4, LDH_D, ME2 631 4 2.575639756 0.188350402 ADHEr, LDH_D, NADH6, PFKand/or FBA and/or TPI 632 4 2.359364668 0.333057496 ADHEr, FUM, LDH_D,NADH6 633 4 2.365271913 0.329105029 ADHEr, HEX1, LDH_D, NADH6 634 42.381170653 0.338701395 ADHEr, LDH_D, NADH6, TAL 635 4 2.4404386210.335394947 ADHEr, LDH_D, NADH6, TKT2 636 4 0.303924042 0.270354967ADHEr, LDH_D, PPCK, TKT2 637 4 0.276184314 0.387717801 ADHEr, HEX1,LDH_D, PPS 638 4 0.158957458 0.24714816 ADHEr, LDH_D, PFK and/or FBAand/or TPI, RPE 639 4 0.268683115 0.271077634 ADHEr, LDH_D, PPCK, RPE640 4 2.268133298 0.177302028 ADHEr, FRD and/or SUCD4, LDH_D, PFK and/orFBA and/or TPI 641 4 2.303972218 0.174839451 ADHEr, FRD and/or SUCD4,LDH_D, PGI 642 4 2.202777355 0.227277897 ADHEr, FRD and/or SUCD4, LDH_D,PPCK 643 4 0.136108259 0.248297219 ADHEr, LDH_D, PFK and/or FBA and/orTPI, TAL 644 4 0.233253283 0.271804175 ADHEr, LDH_D, PPCK, TAL 645 40.753832099 0.262897433 ADHEr, FADH4, LDH_D, THD2 and/or GLUDy 646 41.00803743 0.384504476 ADHEr, ATPS4r, LDH_D, TKT1 647 4 0.7024008770.260125701 ADHEr, FADH4, LDH_D, TKT1 648 4 0.136108259 0.248297219ADHEr, LDH_D, PFK and/or FBA and/or TPI, TKT1 649 4 0.7024008770.260125701 ADHEr, FRD and/or SUCD4, LDH_D, TKT1 650 4 2.3811706530.338701395 ADHEr, LDH_D, NADH6, TKT1 651 4 0.233253283 0.271804175ADHEr, LDH_D, PPCK, TKT1 652 5 0.506864978 0.275350379 ADHEr, ASPT, FUM,LDH_D, TKT2 653 5 2.69303784 0.249064654 ADHEr, ASPT, LDH_D, MDH, TKT2654 5 4.367851715 0.180794605 ADHEr, ASPT, FUM, LDH_D, ME2 655 50.41729748 0.278421057 ADHEr, ASPT, FUM, LDH_D, RPE 656 5 0.3257097580.281560994 ADHEr, ASPT, FUM, LDH_D, TAL 657 5 2.645877173 0.251090969ADHEr, ASPT, LDH_D, MDH, TAL 658 5 0.746129455 0.280922235 ADHEr, ASPT,FUM, LDH_D, THD2 and/or GLUDy 659 5 1.038232852 0.250484522 ADHEr,ATPS4r, FADH4, LDH_D, SUCOAS 660 5 1.091156432 0.260562519 ADHEr,ATPS4r, FADH4, LDH_D, THD2 and/or GLUDy 661 5 3.468709118 0.171072336ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, ME2 662 5 1.6356533310.170872463 ADHEr, ATPS4r, LDH_D, PFK and/or FBA and/or TPI, PFLi 663 50.971889592 0.254878539 ADHEr, ATPS4r, LDH_D, PPCK, PYK 664 52.773151559 0.15594541 ADHEr, ATPS4r, FRD and/or SUCD4, LDH_D, PGI 665 50.794188757 0.220436537 ADHEr, FADH4, HEX1, LDH_D, TKT2 666 54.635305085 0.167733685 ADHEr, FADH4, LDH_D, MDH, TKT2 667 5 0.7262133490.222710178 ADHEr, FADH4, HEX1, LDH_D, RPE 668 5 4.60885325 0.168834272ADHEr, FADH4, LDH_D, MDH, RPE 669 5 4.600497528 0.168070055 ADHEr,FADH4, LDH_D, MDH, SUCOAS 670 5 4.582051994 0.169949398 ADHEr, FADH4,LDH_D, MDH, TAL 671 5 1.218736747 0.252980412 ADHEr, FADH4, LDH_D,SUCOAS, THD2 and/or GLUDy 672 5 2.323324205 0.162516565 ADHEr, LDH_D,PFK and/or FBA and/or TPI, PPCK, TKT2 673 5 0.794188757 0.220436537ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TKT2 674 5 1.080064187 0.261131964ADHEr, FRD and/or SUCD4, ICL, LDH_D, THD2 and/or GLUDy 675 5 1.0800641870.261131964 ADHEr, FRD and/or SUCD4, LDH_D, MALS, THD2 and/or GLUDy 6765 4.635305085 0.167733685 ADHEr, FRD and/or SUCD4, LDH_D, MDH, TKT2 6775 4.611480051 0.167700442 ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE 678 54.603168381 0.166946432 ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS 6795 4.584877196 0.168800591 ADHEr, FRD and/or SUCD4, LDH_D, ME2, TAL 680 54.692124349 0.117711987 ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/orGLUDy 681 5 4.637738386 0.16661454 ADHEr, FRD and/or SUCD4, LDH_D, ME2,TKT2 682 5 4.582051994 0.169949398 ADHEr, FRD and/or SUCD4, LDH_D, MDH,TAL 683 5 1.218736747 0.252980412 ADHEr, FRD and/or SUCD4, LDH_D,SUCOAS, THD2 and/or GLUDy 684 5 2.22459069 0.253337321 ADHEr, FUM,LDH_D, PPCK, TKT2 685 5 2.084553651 0.317680271 ADHEr, FRD and/or SUCD4,FUM, GLU5K, LDH_D 686 5 2.319043241 0.166454441 ADHEr, HEX1, LDH_D, PFKand/or FBA and/or TPI, TKT2 687 5 0.379133409 0.337257295 ADHEr, FUM,HEX1, LDH_D, TKT2 688 5 0.379133409 0.337257295 ADHEr, HEX1, LDH_D, MDH,TKT2 689 5 1.080064187 0.261131964 ADHEr, FADH4, ICL, LDH_D, THD2 and/orGLUDy 690 5 1.080064187 0.261131964 ADHEr, FADH4, LDH_D, MALS, THD2and/or GLUDy 691 5 2.22459069 0.253337321 ADHEr, LDH_D, MDH, PPCK, TKT2692 5 4.611480051 0.167700442 ADHEr, FADH4, LDH_D, ME2, RPE 693 54.603168381 0.166946432 ADHEr, FADH4, LDH_D, ME2, SUCOAS 694 54.584877196 0.168800591 ADHEr, FADH4, LDH_D, ME2, TAL 695 5 4.6921243490.117711987 ADHEr, FADH4, LDH_D, ME2, THD2 and/or GLUDy 696 54.637738386 0.16661454 ADHEr, FADH4, LDH_D, ME2, TKT2 697 5 2.8710648940.268281645 ACKr and/or PTAr, ADHEr, LDH_D, ME2, NADH6 698 5 2.1659248080.255458013 ADHEr, FRD and/or SUCD4, FUM, LDH_D, ME2 699 5 4.3286848550.179395311 ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, ME2 700 51.421860815 0.279607649 ADHEr, FUM, LDH_D, ME2, THD2 and/or GLUDy 701 52.626697246 0.185479991 ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI,TKT2 702 5 2.515320971 0.228708985 ADHEr, FUM, LDH_D, NADH6, PPCK 703 52.3870222 0.31455217 ADHEr, FUM, HEX1, LDH_D, NADH6 704 5 2.4527544210.325482265 ADHEr, HEX1, LDH_D, NADH6, TKT2 705 5 2.5153209710.228708985 ADHEr, LDH_D, MDH, NADH6, PPCK 706 5 2.517581576 0.273304492ADHEr, LDH_D, MDH, NADH6, TKT2 707 5 2.44490357 0.275824433 ADHEr, FUM,LDH_D, ME2, NADH6 708 5 2.6103794 0.186397367 ADHEr, LDH_D, NADH6, PFKand/or FBA and/or TPI, RPE 709 5 2.555986853 0.242393036 ADHEr, LDH_D,NADH6, PPCK, TKT2 710 5 2.421064631 0.329399782 ADHEr, FUM, LDH_D,NADH6, RPE 711 5 2.424713593 0.326643472 ADHEr, HEX1, LDH_D, NADH6, RPE712 5 2.535155189 0.243219368 ADHEr, LDH_D, NADH6, PPCK, RPE 713 52.593899337 0.187323863 ADHEr, LDH_D, NADH6, PFK and/or FBA and/or TPI,TAL 714 5 2.391804455 0.331134392 ADHEr, FUM, LDH_D, NADH6, TAL 715 52.470792173 0.274926806 ADHEr, LDH_D, MDH, NADH6, TAL 716 5 2.5141810060.244051353 ADHEr, LDH_D, NADH6, PPCK, TAL 717 5 0.318612958 0.180159999ACKr and/or PTAr, ADHEr, LDH_D, MDH, PFK and/or FBA and/or TPI 718 52.298067641 0.164222738 ADHEr, LDH_D, PFK and/or FBA and/or TPI, PPCK,TAL 719 5 1.630847318 0.197351873 ADHEr, LDH_D, MDH, PFK and/or FBAand/or TPI, PFLi 720 5 4.068156517 0.116307981 ADHEr, LDH_D, PFK and/orFBA and/or TPI, PFLi, PPCK 721 5 1.630847318 0.197351873 ADHEr, FUM,LDH_D, PFK and/or FBA and/or TPI, PFLi 722 5 1.732749142 0.29901565ADHEr, FUM, HEX1, LDH_D, PFLi 723 5 1.717432965 0.229711682 ADHEr, HEX1,LDH_D, PFLi, PPCK 724 5 3.998783556 0.193038233 ADHEr, FRD and/or SUCD4,LDH_D, ME2, PFLi 725 5 4.075902556 0.129526478 ADHEr, LDH_D, NADH6, PFKand/or FBA and/or TPI, PFLi 726 5 3.837085522 0.275765851 ADHEr, LDH_D,NADH6, PFLi, RPE 727 5 3.820196202 0.277060587 ADHEr, LDH_D, NADH6,PFLi, TAL 728 5 3.853817724 0.274483161 ADHEr, LDH_D, NADH6, PFLi, TKT2729 5 3.839632897 0.261974412 ADHEr, FRD and/or SUCD4, LDH_D, PFLi, THD2and/or GLUDy 730 5 4.393030474 0.140852085 ADHEr, EDA and/or PGDHY, FRDand/or SUCD4, LDH_D, PGI 731 5 0.430495607 0.382946425 ADHEr, HEX1,LDH_D, PPS, TKT2 732 5 0.381078242 0.384474433 ADHEr, HEX1, LDH_D, PPS,RPE 733 5 0.331264934 0.386014683 ADHEr, HEX1, LDH_D, PPS, TAL 734 50.331264934 0.386014683 ADHEr, HEX1, LDH_D, PPS, TKT1 735 5 0.3186129580.180159999 ACKr and/or PTAr, ADHEr, FUM, LDH_D, PFK and/or FBA and/orTPI 736 5 2.310761865 0.163365197 ADHEr, LDH_D, PFK and/or FBA and/orTPI, PPCK, RPE 737 5 2.306254619 0.167256205 ADHEr, HEX1, LDH_D, PFKand/or FBA and/or TPI, RPE 738 5 0.335428873 0.338418234 ADHEr, HEX1,LDH_D, MDH, RPE 739 5 2.674115155 0.222196435 ADHEr, ASPT, FRD and/orSUCD4, LDH_D, MDH 740 5 2.310408468 0.174397201 ADHEr, FRD and/or SUCD4,LDH_D, PFK and/or FBA and/or TPI, TKT2 741 5 2.084492611 0.317726946ADHEr, ASNS2, FRD and/or SUCD4, FUM, LDH_D 742 5 2.086878053 0.315902866ADHEr, CBMK2, FRD and/or SUCD4, FUM, LDH_D 743 5 2.084553651 0.317680271ADHEr, FRD and/or SUCD4, FUM, G5SD, LDH_D 744 5 2.221671127 0.212830356ADHEr, FRD and/or SUCD4, FUM, LDH_D, PPCK 745 5 2.110766092 0.297636347ADHEr, FRD and/or SUCD4, FUM, HEX1, LDH_D 746 5 2.221671127 0.212830356ADHEr, FRD and/or SUCD4, LDH_D, MDH, PPCK 747 5 2.29690811 0.175324843ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBA and/or TPI, RPE 748 52.283263365 0.176262405 ADHEr, FRD and/or SUCD4, LDH_D, PFK and/or FBAand/or TPI, TAL 749 5 2.799273371 0.150483141 ACKr and/or PTAr, ADHEr,FRD and/or SUCD4, LDH_D, PGI 750 5 2.255979533 0.224464061 ADHEr, FRDand/or SUCD4, LDH_D, PPCK, TKT2 751 5 4.330026614 0.179036754 ACKrand/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, MDH 752 5 2.2389426910.225365131 ADHEr, FRD and/or SUCD4, LDH_D, PPCK, RPE 753 5 2.1199389590.309086971 ADHEr, FRD and/or SUCD4, HEX1, LDH_D, TAL 754 5 2.2217685170.226273464 ADHEr, FRD and/or SUCD4, LDH_D, PPCK, TAL 755 5 2.1865458130.254918542 ADHEr, FUM, LDH_D, PPCK, TAL 756 5 2.293342202 0.16806573ADHEr, HEX1, LDH_D, PFK and/or FBA and/or TPI, TAL 757 5 0.291422410.339587193 ADHEr, FUM, HEX1, LDH_D, TAL 758 5 0.29142241 0.339587193ADHEr, HEX1, LDH_D, MDH, TAL 759 5 2.186545813 0.254918542 ADHEr, LDH_D,MDH, PPCK, TAL 760 5 3.603753429 0.189374217 ADHEr, FADH4, HEX1, LDH_D,THD2 and/or GLUDy 761 5 4.688321717 0.11916587 ADHEr, FADH4, LDH_D, MDH,THD2 and/or GLUDy 762 5 3.879826068 0.057433556 ADHEr, FADH4, LDH_D,PPCK, THD2 and/or GLUDy 763 5 2.092923946 0.31127974 ADHEr, FUM, HEX1,LDH_D, THD2 and/or GLUDy 764 5 1.181362879 0.252361787 ADHEr, HEX1,LDH_D, PPCK, THD2 and/or GLUDy 765 5 0.325709758 0.281560994 ADHEr,ASPT, FUM, LDH_D, TKT1 766 5 2.645877173 0.251090969 ADHEr, ASPT, LDH_D,MDH, TKT1 767 5 4.582051994 0.169949398 ADHEr, FADH4, LDH_D, MDH, TKT1768 5 2.298067641 0.164222738 ADHEr, LDH_D, PFK and/or FBA and/or TPI,PPCK, TKT1 769 5 4.582051994 0.169949398 ADHEr, FRD and/or SUCD4, LDH_D,MDH, TKT1 770 5 4.584877196 0.168800591 ADHEr, FRD and/or SUCD4, LDH_D,ME2, TKT1 771 5 2.186545813 0.254918542 ADHEr, FUM, LDH_D, PPCK, TKT1772 5 2.293342202 0.16806573 ADHEr, HEX1, LDH_D, PFK and/or FBA and/orTPI, TKT1 773 5 0.29142241 0.339587193 ADHEr, FUM, HEX1, LDH_D, TKT1 7745 0.29142241 0.339587193 ADHEr, HEX1, LDH_D, MDH, TKT1 775 5 2.1865458130.254918542 ADHEr, LDH_D, MDH, PPCK, TKT1 776 5 4.584877196 0.168800591ADHEr, FADH4, LDH_D, ME2, TKT1 777 5 2.593899337 0.187323863 ADHEr,LDH_D, NADH6, PFK and/or FBA and/or TPI, TKT1 778 5 2.3918044550.331134392 ADHEr, FUM, LDH_D, NADH6, TKT1 779 5 2.396471968 0.327812994ADHEr, HEX1, LDH_D, NADH6, TKT1 780 5 2.470792173 0.274926806 ADHEr,LDH_D, MDH, NADH6, TKT1 781 5 2.514181006 0.244051353 ADHEr, LDH_D,NADH6, PPCK, TKT1 782 5 3.820196202 0.277060587 ADHEr, LDH_D, NADH6,PFLi, TKT1 783 5 2.283263365 0.176262405 ADHEr, FRD and/or SUCD4, LDH D,PFK and/or FBA and/or TPI, TKT1 784 5 2.119938959 0.309086971 ADHEr, FRDand/or SUCD4, HEX1, LDH_D, TKT1 785 5 2.221768517 0.226273464 ADHEr, FRDand/or SUCD4, LDH_D, PPCK, TKT1 786 6 4.63815309 0.138347146 ADHEr,ASPT, FADH4, LDH_D, MDH, PYK 787 6 4.459389393 0.176944819 ADHEr, ASPT,FUM, LDH_D, MDH, TKT2 788 6 4.380192095 0.177265253 ADHEr, ASPT, FRDand/or SUCD4, LDH_D, MALS, MDH 789 6 4.40071793 0.179412355 ADHEr, ASPT,LDH_D, MALS, MDH, TAL 790 6 4.60130813 0.167781976 ADHEr, ASPT, LDH_D,MDH, SUCOAS, TKT2 791 6 4.459389393 0.176944819 ADHEr, ASPT, FUM, LDH_D,ME2, TKT2 792 6 4.430256792 0.178170044 ADHEr, ASPT, FUM, LDH_D, ME2,RPE 793 6 4.40071793 0.179412355 ADHEr, ASPT, FUM, LDH_D, ME2, TAL 794 64.563806878 0.124751372 ADHEr, ASPT, FUM, LDH_D, ME2, THD2 and/or GLUDy795 6 4.380192095 0.177265253 ADHEr, ASPT, FRD and/or SUCD4, FUM, LDH_D,MDH 796 6 4.380192095 0.177265253 ADHEr, ASPT, FRD and/or SUCD4, FUM,LDH_D, ME2 797 6 4.40071793 0.179412355 ADHEr, ASPT, FUM, LDH_D, MDH,TAL 798 6 4.547590285 0.169998972 ADHEr, ASPT, LDH_D, MDH, SUCOAS, TAL799 6 4.32598859 0.179624623 ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH,THD2 and/or GLUDy 800 6 4.503409516 0.124027245 ADHEr, ASPT, ATPS4r,LDH_D, MDH, TKT2 801 6 4.55209485 0.121669936 ADHEr, ASPT, ATPS4r,LDH_D, MDH, PGDH 802 6 4.519337222 0.123256037 ADHEr, ASPT, ATPS4r,LDH_D, MDH, RPE 803 6 4.535068073 0.12249436 ADHEr, ASPT, ATPS4r, LDH_D,MDH, TAL 804 6 5.189612621 0.143792027 ADHEr, ATPS4r, FRD and/or SUCD4,LDH_D, ME2, PFLi 805 6 4.321178853 0.120168187 ADHEr, ATPS4r, FRD and/orSUCD4, LDH_D, MDH, PGI 806 6 4.555303308 0.170023606 ADHEr, DAAD, FRDand/or SUCD4, LDH_D, ME2, PRO1z 807 6 4.552254757 0.171189177 ADHEr,DAAD, FRD and/or SUCD4, LDH_D, MDH, PRO1z 808 6 4.611166872 0.137118286ADHEr, FADH4, FUM, LDH_D, MDH, PYK 809 6 4.681108251 0.164738111 ADHEr,FADH4, LDH_D, MDH, SUCOAS, TKT2 810 6 4.655427003 0.165799614 ADHEr,FADH4, LDH_D, MDH, RPE, SUCOAS 811 6 4.62941265 0.166874885 ADHEr,FADH4, LDH_D, MDH, SUCOAS, TAL 812 6 4.57471951 0.178914826 ADHEr,FADH4, HEX1, LDH_D, SUCOAS, THD2 and/or GLUDy 813 6 4.721400480.117206382 ADHEr, FADH4, LDH_D, MDH, SUCOAS, THD2 and/or GLUDy 814 65.77503303 0.099302315 ADHEr, EDA and/or PGDHY, FADH4, LDH_D, MDH, THD2and/or GLUDy 815 6 4.802404267 0.049107371 ACKr and/or PTAr, ADHEr,FADH4, LDH_D, PPCK, THD2 and/or GLUDy 816 6 4.611166872 0.137118286ADHEr, FRD and/or SUCD4, FUM, LDH_D, MDH, PYK 817 6 4.6811082510.164738111 ADHEr, FRD and/or SUCD4, LDH_D, MDH, SUCOAS, TKT2 818 64.640728933 0.137362308 ADHEr, FRD and/or SUCD4, LDH_D, MDH, ME2, PYK819 6 4.657699713 0.164706045 ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE,SUCOAS 820 6 4.683198197 0.163658455 ADHEr, FRD and/or SUCD4, LDH_D,ME2, SUCOAS, TKT2 821 6 4.63187269 0.165767133 ADHEr, FRD and/or SUCD4,LDH_D, ME2, SUCOAS, TAL 822 6 5.443882794 0.111598427 ADHEr, FRD and/orSUCD4, HEX1, LDH_D, ME2, THD2 and/or GLUDy 823 6 4.714403301 0.10919394ACKr and/or PTAr, ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/or GLUDy824 6 4.727966085 0.117420509 ADHEr, FRD and/or SUCD4, LDH_D, ME2, RPE,THD2 and/or GLUDy 825 6 4.724744313 0.115799637 ADHEr, FRD and/or SUCD4,LDH_D, ME2, SUCOAS, THD2 and/or GLUDy 826 6 4.710892193 0.11755936ADHEr, FRD and/or SUCD4, LDH_D, ME2, TAL, THD2 and/or GLUDy 827 64.744999691 0.117281986 ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2 and/orGLUDy, TKT2 828 6 4.62941265 0.166874885 ADHEr, FRD and/or SUCD4, LDH_D,MDH, SUCOAS, TAL 829 6 4.74186178 0.118725202 ADHEr, FRD and/or SUCD4,LDH_D, MDH, THD2 and/or GLUDy, TKT2 830 6 4.512318137 0.052959973 ADHEr,FRD and/or SUCD4, LDH_D, PPCK, PYK, THD2 and/or GLUDy 831 6 4.7246145640.118867157 ADHEr, FRD and/or SUCD4, LDH_D, MDH, RPE, THD2 and/or GLUDy832 6 4.707326054 0.119009453 ADHEr, FRD and/or SUCD4, LDH_D, MDH, TAL,THD2 and/or GLUDy 833 6 4.459389393 0.176944819 ADHEr, ASPT, ICL, LDH_D,MDH, TKT2 834 6 4.380192095 0.177265253 ADHEr, ASPT, FRD and/or SUCD4,ICL, LDH_D, MDH 835 6 4.40071793 0.179412355 ADHEr, ASPT, ICL, LDH_D,MDH, TAL 836 6 4.874048363 0.048155873 ADHEr, FADH4, ICL, LDH_D, PPCK,THD2 and/or GLUDy 837 6 4.520685593 0.183259208 ADHEr, FADH4, HEX1, ICL,LDH_D, THD2 and/or GLUDy 838 6 4.459389393 0.176944819 ADHEr, ASPT,LDH_D, MALS, MDH, TKT2 839 6 4.520685593 0.183259208 ADHEr, FADH4, HEX1,LDH_D, MALS, THD2 and/or GLUDy 840 6 4.874048363 0.048155873 ADHEr,FADH4, LDH_D, MALS, PPCK, THD2 and/or GLUDy 841 6 4.5175328210.171239478 ADHEr, ASPT, FUM, LDH_D, ME2, SUCOAS 842 6 4.6407289330.137362308 ADHEr, FADH4, LDH_D, MDH, ME2, PYK 843 6 4.6576997130.164706045 ADHEr, FADH4, LDH_D, ME2, RPE, SUCOAS 844 6 4.6831981970.163658455 ADHEr, FADH4, LDH_D, ME2, SUCOAS, TKT2 845 6 4.631872690.165767133 ADHEr, FADH4, LDH_D, ME2, SUCOAS, TAL 846 6 4.7247443130.115799637 ADHEr, FADH4, LDH_D, ME2, SUCOAS, THD2 and/or GLUDy 847 64.744999691 0.117281986 ADHEr, FADH4, LDH_D, ME2, THD2 and/or GLUDy,TKT2 848 6 4.526462669 0.168070055 ADHEr, FRD and/or SUCD4, FUM, LDH_D,ME2, SUCOAS 849 6 5.443882794 0.111598427 ADHEr, FADH4, HEX1, LDH_D,ME2, THD2 and/or GLUDy 850 6 4.714403301 0.10919394 ACKr and/or PTAr,ADHEr, FADH4, LDH_D, ME2, THD2 and/or GLUDy 851 6 4.7279660850.117420509 ADHEr, FADH4, LDH_D, ME2, RPE, THD2 and/or GLUDy 852 64.710892193 0.11755936 ADHEr, FADH4, LDH_D, ME2, TAL, THD2 and/or GLUDy853 6 4.33071542 0.180983121 ACKr and/or PTAr, ADHEr, ASPT, LDH_D, MDH,NADH6 854 6 4.313819364 0.203396482 ACKr and/or PTAr, ADHEr, LDH_D, MDH,NADH6, THD2 and/or GLUDy 855 6 4.836109146 0.120863498 ADHEr, FRD and/orSUCD4, LDH_D, ME2, PFLi, PGI 856 6 5.044438059 0.165184738 ACKr and/orPTAr, ADHEr, HEX1, LDH_D, PFLi, PGI 857 6 5.168015107 0.134224131 ADHEr,FRD and/or SUCD4, LDH_D, ME2, PFLi, THD2 and/or GLUDy 858 6 5.3204558270.059323173 ADHEr, FRD and/or SUCD4, LDH_D, MDH, PFLi, THD2 and/or GLUDy859 6 4.74186178 0.118725202 ADHEr, FADH4, LDH_D, MDH, THD2 and/orGLUDy, TKT2 860 6 4.512318137 0.052959973 ADHEr, FADH4, LDH_D, PPCK,PYK, THD2 and/or GLUDy 861 6 4.724614564 0.118867157 ADHEr, FADH4,LDH_D, MDH, RPE, THD2 and/or GLUDy 862 6 4.888461627 0.046924032 ADHEr,FADH4, LDH_D, PPCK, SUCOAS, THD2 and/or GLUDy 863 6 4.7073260540.119009453 ADHEr, FADH4, LDH_D, MDH, TAL, THD2 and/or GLUDy 864 64.40071793 0.179412355 ADHEr, ASPT, FUM, LDH_D, MDH, TKT1 865 64.547590285 0.169998972 ADHEr, ASPT, LDH_D, MDH, SUCOAS, TKT1 866 64.535068073 0.12249436 ADHEr, ASPT, ATPS4r, LDH_D, MDH, TKT1 867 64.62941265 0.166874885 ADHEr, FADH4, LDH_D, MDH, SUCOAS, TKT1 868 64.63187269 0.165767133 ADHEr, FRD and/or SUCD4, LDH_D, ME2, SUCOAS, TKT1869 6 4.710892193 0.11755936 ADHEr, FRD and/or SUCD4, LDH_D, ME2, THD2and/or GLUDy, TKT1 870 6 4.62941265 0.166874885 ADHEr, FRD and/or SUCD4,LDH_D, MDH, SUCOAS, TKT1 871 6 4.707326054 0.119009453 ADHEr, FRD and/orSUCD4, LDH_D, MDH, THD2 and/or GLUDy, TKT1 872 6 4.40071793 0.179412355ADHEr, ASPT, ICL, LDH_D, MDH, TKT1 873 6 4.40071793 0.179412355 ADHEr,ASPT, LDH_D, MALS, MDH, TKT1 874 6 4.40071793 0.179412355 ADHEr, ASPT,FUM, LDH_D, ME2, TKT1 875 6 4.63187269 0.165767133 ADHEr, FADH4, LDH_D,ME2, SUCOAS, TKT1 876 6 4.710892193 0.11755936 ADHEr, FADH4, LDH_D, ME2,THD2 and/or GLUDy, TKT1 877 6 4.707326054 0.119009453 ADHEr, FADH4,LDH_D, MDH, THD2 and/or GLUDy, TKT1

Minimal Gene Deletion Sets for Enhanced Production of 6-ACA.

Described above are strain design strategies for improving6-aminocaproate (6-ACA) production in microorganisms that possess the6-ACA pathway via acetyl-CoA and succinyl-CoA. Based on an extensiveanalysis of the strain designs for 6-ACA production described in Table14, a minimum set of deletions that is required for growth-coupled 6-ACAproduction has been identified. Note that phosphoenolpyruvatecarboxykinase (PPCK) was assumed to be reversible.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactatedehydrogenase (LDH_D) are required for preventing the formation ofcompeting byproducts, ethanol and lactate. Therefore, the minimaldeletion set comprises deletion of acetaldehyde dehydrogenase (ADHEr)and lactate dehydrogenase (LDH_D). Additional deletion strains includestrains lacking at least one of the following activities in addition toADHEr and LDH_D: malate dehydrogenase (MDH), aspartase (ASPT), NAD(P)transhydrogenase (THD2), and glutamate dehydrogenase (GLUDy). Suchadditional deletions result in a much tighter coupling of productionwith cell growth. FIGS. 28-31 show calculated 6-ACA versus growth yieldsfor deletion mutants having at least deletion of ADHEr and LDH_D (FIG.28). Calculated yields for strains with additional deletions are shownin FIGS. 29-31.

An additional minimal set of deletions includes phosphoglucoisomerase(PGI). This design focuses on generating reducing equivalents via thepentose phosphate pathway. Additional beneficial deletions include anyof: acetaldehyde dehydrogenase (ADHEr), hexokinase (HEX1),2-dehydro-3-deoxy-phosphogluconate aldolase (EDA) and phosphogluconatedehydratase (PGDHy). FIGS. 32-34 show calculated 6-ACA versus growthyields for deletion mutants having at least deletion of PGI, withadditional exemplary mutants shown FIGS. 32-34.

Each of these strains can be supplemented with additional deletions ifit is determined that the predicted strain designs do not sufficientlycouple the formation of the product with biomass formation or forincreased efficiency of coupling the formation of the product withbiomass formation. Alternatively, some other enzymes not known topossess significant activity under the growth conditions can becomeactive due to adaptive evolution or random mutagenesis. Such enzymeactivities can also be knocked out. For example, succinate dehydrogenasethat oxidizes succinate to fumarate and is known to be active only underaerobic conditions can assume significant activity even under anaerobicconditions, and therefore such activity can be knocked out. However, thelist of gene deletion sets provided herein serves as a good startingpoint for construction of high-yielding growth-coupled 6-ACA productionstrains.

Minimal Gene Deletion Sets for Enhanced Production of Adipate.

Described above are strain design strategies for improving adipateproduction in microorganisms that possess the adipate pathway viaacetyl-CoA and succinyl-CoA. Based on an extensive analysis of thestrain designs for adipate production described in Table 15, a minimumset of deletions that is required for growth-coupled adipate productionin the network has been identified. Note that phosphoenolpyruvatecarboxykinase (PPCK) was assumed to be reversible in the network.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactatedehydrogenase (LDH_D) are required for preventing the formation ofcompeting byproducts, ethanol and lactate. Therefore, the minimaldeletion set comprises deletion of acetaldehyde dehydrogenase (ADHEr)and lactate dehydrogenase (LDH_D). Additional deletion strains include astrain lacking at least one of the following activities in addition toacetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D):fumarase (FUM), phosphoglucose isomerase (PGI), PEP carboxykinase(PPCK), hexokinase (HEX1), malate dehydrogenase (MDH), and NADHdehydrogenase (NADH6).

Additional deletions have been identified by the OptKnock framework forimproving the growth-coupled formation of adipate. These include one ormore of the following: malic enzyme (ME2), aspartate transaminase(ASPT), acetate kinase (ACKr), phosphotransacetylase (PTAr), pyruvateformate lyase (PFL), transhydrogenase (THD2), and glutamatedehydrogenase (GLUDy), and PTS system of glucose uptake (GLCpts).Further improvements in yields can be achieved by additional deletionsin any of the following enzymes: ATP synthase (ATPS4r), phosphogluconatedehydratase (PGDHY), 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA),6-phosphogluconolactonase (PGL), glucose 6-phosphate dehydrogenase(G6PDHY), and phosphogluconate dehydrogenase (PGDH).

Each of these strains can be supplemented with additional deletions ifit is determined that the predicted strain designs do not sufficientlycouple the formation of the product with biomass formation or forincreased efficiency of coupling the formation of the product withbiomass formation. Alternatively, some other enzymes not known topossess significant activity under the growth conditions can becomeactive due to adaptive evolution or random mutagenesis. Such enzymeactivities can also be knocked out. However, the list of gene deletionsets provided herein serves as a good starting point for construction ofhigh-yielding growth-coupled adipate production strains.

Minimal Gene Deletion Sets for Enhanced Production of HMDA.

Described above are strain design strategies for improving hexamethylenediamine (HMDA) production in microorganisms that possess the HMDApathway via acetyl-CoA and succinyl-CoA. Based on an extensive analysisof the strain designs for HMDA production described in Table 16, aminimum set of deletions that is required for growth-coupled HMDAproduction in the network has been identified. Note thatphosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible inthe network.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactatedehydrogenase (LDH_D) are required for preventing the formation ofcompeting byproducts, ethanol and lactate. Therefore, the minimaldeletion set comprises deletion of acetaldehyde dehydrogenase (ADHEr)and lactate dehydrogenase (LDH_D). Additional deletion strains include astrain lacking at least one of the following activities in addition toacetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D):fumarate reductase (FRD2), fumarase (FUM), phosphoglucose isomerase(PGI), or PEP carboxykinase (PPCK).

Additional deletions have been identified by the OptKnock framework forimproving the growth-coupled formation of HMDA. These include one ormore of the following: hexokinase (HEX1), malic enzyme (ME2), malatedehydrogenase (MDH), aspartate transaminase (ASPT), acetate kinase(ACKr), phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), andpyruvate kinase (PYK). The HMDA yields can be further improved byfurther deleting one or more of the following enzymes: transhydrogenase(THD2), glutamate dehydrogenase (GLUDy), ATP synthase (ATPS4r), GLCpts(PTS system of glucose uptake), PGDHY (phosphogluconate dehydratase) andEDA (2-dehydro-3-deoxy-phosphogluconate aldolase).

Each of these strains can be supplemented with additional deletions ifit is determined that the predicted strain designs do not sufficientlycouple the formation of the product with biomass formation or forincreased efficiency of coupling the formation of the product withbiomass formation. Alternatively, some other enzymes not known topossess significant activity under the growth conditions can becomeactive due to adaptive evolution or random mutagenesis. Such enzymeactivities can also be knocked out. For example, succinate dehydrogenasethat oxidizes succinate to fumarate and is known to be active only underaerobic conditions can assume significant activity even under anaerobicconditions, and therefore such activity can be knocked out. However, thelist of gene deletion sets provided herein serves as a good startingpoint for construction of high-yielding growth-coupled HMDA productionstrains.

Optknock Strain Design for Growth-Coupled Production of Adipic Acid.

Described below is a further exemplification of a deletion strategy forengineering a strain to synthesize adipate using a succinyl-CoA pathway.All high-priority growth-coupled designs for adipate synthesis buildupon a strain lacking acetylaldehyde-CoA dehydrogenase (ADHEr) andlactate dehydrogenase (LDH_D) activities to prevent the formation offermentation byprouducts, as discussed above. The further deletion ofmalate dehydrogenase (MDH) also reduces byproduct production. FIG. 35shows growth-coupled adipate production characteristics of high prioritystrain designs (grey) compared with that of wild-type E. coli (black). Aglucose uptake rate of 10 mmol/gDW/hr is assumed. A strain deficient inADHEr, LDH_D and MDH activities (Design 1 in FIG. 35) is predicted toachieve an adipate yield of 0.51 grams adipate per gram of glucoseutilized (g/g) at the maximum biomass yield.

Designs 2-4 build on Design 1 as a base design. Design 2 entails theremoval of phosphoenolpyruvate carboxykinase (PPCK). This designimproves the adipate yield to 3.6 g/g at the maximum biomass yield. Theadditional deletion of pyruvate formate lyase (PFLi) activity in Design3 further improves yield by preventing secretion of formate as abyproduct. The predicted adipate yield of this design is 5.8 g/g. Design4 features the deletion of NAD(P) transhydrogenase (THD2) in addition toADHEr, LDH_D, MDH, PPCK and PFLi. This results in an adipate yield of6.8 g/g at a growth rate of 0.117 l/hr. Design 4 serves to tightlycouple adipate production to cell growth while achieving 91% of thetheoretical maximum yield.

Example XXXI Biosynthesis of Adipate Semialdehyde from Adipate and6-Aminocaproate Semialdehyde from 6-Aminocaproate

This Example describes the biosynthetic production of adipatesemialdehyde from adipate and of 6-aminocaproate semialdehyde from6-aminocaproate.

The transformation from adipate to adipate semialdehyde (FIG. 25, stepX) can by catalyzed by the carboxylic acid reductases (CAR). This isdemonstrated by the following results. Chemically competent cells of E.coli strain ECKh-422 (ΔadhE, ΔldhA, ΔpflB, ΔlpdA, integrated lpdA fromKlebsiella pneumonia::E354K, Δmdh, ΔarcA, gltA-R163L) with f′pKLJ33swere transformed with pZs*13s plasmids harboring various CAR gene (Table17) or a control plasmid without any CAR gene. Single colonies of thetransformations were selected and grown overnight in LB at 37° C. with100 μg/ml carbenecillin and 10 μg/ml chloramphenicol. The cells weresubcultured at a ratio of 1:50 and induced with 200 μM IPTG at an OD600of 0.6. Cells were incubated for 5 hours at 37° C. before harvesting.Cell cultures were aliquoted into 15 ml samples and pelleted. The cellpellets were stored at −80° C. until used for the assay.

TABLE 17 CAR genes used this example Protein GenBank ID GI NumberOrganism 720 AAR91681.1 40796035 Nocardia iowensis 889 YP_887275.1118473501 Mycobacterium smegmatis str. MC2 155 890 YP_889972.1 118469671Mycobacterium smegmatis str. MC2 155 891 NP_959974.1 41407138Mycobacterium avium subsp. paratuberculosis K-10 892 YP_001850422.1183982131 Mycobacterium marinum M

Cell pellets were lysed by addition of 500 μl B-PER with 0.5 μl lysozymeand benzonase. CAR activity was measured by addition of 2 μl of crudelysate to the assay solution of 50 mM Tris (pH 7.2), 1 mM EDTA, 10 mMMgCl₂, 1 mM DTT, 10% (v/v) glycerol, 1 mM ATP, 0.5 mM NADPH and 20 mMAdipate or 50 mM 6-Aminocaproate in a 96 well format microplate with atotal volume of 250 μl. The oxidation of NADPH to NADP⁺ was monitored atan absorbance of 340 nm for 30 minutes at room temperature. The rate ofNADPH depletion was used to calculate activity of the various CARproteins. Total protein concentration of each lysate was determined byBradford, and the activity was normalized to the total proteinconcentration (Units/mg).

Transformation from Adipate to Adipate Semialdehyde (FIG. 24, Step).

As shown in FIG. 36, significant CAR activities using adipate as asubstrate was observed with both CAR genes 889 and 891, whereas thecontrol lysate did not show CAR activity.

Furthermore, a reaction was setup consisting of 500 μl of 50 mM Tris (pH7.2), 1 mM EDTA, 10 mM MgCl₂, 1 mM DTT, 10% (v/v) glycerol, 5 mM ATP, 3mM NADPH and 20 mM Adipate. The reaction was incubated at roomtemperature for 30 minutes and stopped by addition of 1% formic acid.The sample was then centrifuged and the supernatant was analyzed byLC-MS. Low mM level of adipate semialdehyde was detected that confirmedthe transformation from adipate to adipate semialdehyde.

Transformation from 6-aminocaproate to 6-aminocaproate Semialdehyde.

As shown in FIG. 37, significant CAR activities using 6-aminocaproate asa substrate was observed with several CAR genes 720, 889, 890, 891 and892, whereas the control lysate did not show CAR activity. These resultsindicate the transformation from 6-aminocaproate to 6-aminocaproatesemialdehyde.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1-94. (canceled)
 95. A non-naturally occurring microbial organism,comprising a microbial organism having a 6-aminocaproic acid (6-ACA)pathway comprising at least one exogenous nucleic acid encoding a 6-ACApathway enzyme expressed in a sufficient amount to produce 6-ACA, said6-ACA pathway comprising a 4-hydroxy-2-oxoheptane-1,7-dioate (HODH)aldolase, a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, a2-oxohept-4-ene-1,7-dioate (OHED) reductase, a 2-oxoheptane-1,7-dioate(2-OHD) decarboxylase, an adipate semialdehyde aminotransferase, anadipate semialdehyde oxidoreductase (aminating), a2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, a 6-oxohex-4-enoate(6-OHE) reductase, a 2-oxoheptane-1,7-dioate (2-OHD) aminotransferase, a2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (aminating), a2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase, a2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (aminating), a2-aminohept-4-ene-1,7-dioate (2-AHE) reductase, a4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate-lyase, a4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, a3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA reductase, aadipyl-CoA dehydrogenase, a 2-oxohept-4-ene-1,7-dioate (OHED)formate-lyase, a 2-oxohept-4-ene-1,7-dioate (OHED) dehydrogenase, a2-oxoheptane-1,7-dioate (2-OHD) formate-lyase, a 2-oxoheptane-1,7-dioate(2-OHD) dehydrogenase, or a pyruvate formate-lyase activating enzyme.96. The non-naturally occurring microbial organism of claim 95, whereinsaid microbial organism comprises at least one exogenous nucleic acideach encoding a 6-ACA pathway enzyme, said 6-ACA pathway comprising apathway selected from the group consisting of: (i) an HODH aldolase; anOHED hydratase; an OHED reductase; a 2-OHD decarboxylase; or an adipatesemialdehyde aminotransferase or an adipate semialdehyde oxidoreductase(aminating); (ii) an HODH aldolase; an OHED hydratase; an OHEDdecarboxylase; a 6-OHE reductase; or an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating);(iii) an HODH aldolase; an OHED hydratase; an OHED aminotransferase oran OHED oxidoreductase (aminating); a 2-AHE reductase; or a 2-AHDdecarboxylase; (iv) an HODH aldolase; an OHED hydratase; an OHEDreductase; a 2-OHD aminotransferase or a 2-OHD oxidoreductase(aminating); or a 2-AHD decarboxylase; (v) an HODH aldolase; an HODHformate-lyase and a pyruvate formate-lyase activating enzyme or an HODHdehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating); (vi) an HODH aldolase; an OHED hydratase; anOHED formate-lyase and a pyruvate formate-lyase activating enzyme orOHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoAdehydrogenase; or an adipate semialdehyde aminotransferase or an adipatesemialdehyde oxidoreductase (aminating); and (vii) an HODH aldolase; anOHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvateformate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoAdehydrogenase; or an adipate semialdehyde aminotransferase or an adipatesemialdehyde oxidoreductase (aminating). 97-102. (canceled)
 103. Thenon-naturally occurring microbial organism of claim 95, wherein saidmicrobial organism comprises a set of exogenous nucleic acids encoding6-ACA pathway enzymes, said set of exogenous nucleic acids encoding: (i)an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHDdecarboxylase; and an adipate semialdehyde aminotransferase or anadipate semialdehyde oxidoreductase (aminating); (ii) an HODH aldolase;an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase; and anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating); (iii) an HODH aldolase; an OHED hydratase;an OHED aminotransferase or an OHED oxidoreductase (aminating); a 2-AHEreductase; and a 2-AHD decarboxylase; (iv) an HODH aldolase; an OHEDhydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHDoxidoreductase (aminating); and a 2-AHD decarboxylase; (v) an HODHaldolase; an HODH formate-lyase and a pyruvate formate-lyase activatingenzyme or an HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and anadipate semialdehyde aminotransferase or an adipate semialdehydeoxidoreductase (aminating); (vi) an HODH aldolase; an OHED hydratase; anOHED formate-lyase and a pyruvate formate-lyase activating enzyme orOHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoAdehydrogenase; and an adipate semialdehyde aminotransferase or anadipate semialdehyde oxidoreductase (aminating); or (vii) an HODHaldolase; an OHED hydratase; an OHED reductase; a 2-OHD formate-lyaseand a pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase;an adipyl-CoA dehydrogenase; and an adipate semialdehydeaminotransferase or an adipate semialdehyde oxidoreductase (aminating).104-134. (canceled)
 135. A non-naturally occurring microbial organism,comprising a microbial organism having a hexamethylenediamine (HMDA)pathway comprising at least one exogenous nucleic acid encoding a HMDApathway enzyme expressed in a sufficient amount to produce HMDA, saidHMDA pathway comprising a glutamyl-CoA transferase, a glutamyl-CoAligase, a beta-ketothiolase, an 3-oxo-6-aminopimeloyl-CoAoxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase, a6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoAreductase (aldehyde forming), a 2-amino-7-oxoheptanoateaminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, ora homolysine decarboxylase. 136-142. (canceled)
 143. The non-naturallyoccurring microbial organism of claim 135, wherein said microbialorganism comprises a set of exogenous nucleic acids encoding HMDApathway enzymes expressed in a sufficient amount to produce HMDA, saidset of exogenous nucleic acids encoding a glutamyl-CoA transferase or aglutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoAoxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferaseor aminating oxidoreductase; and a homolysine decarboxylase. 144-145.(canceled)
 146. A non-naturally occurring microbial organism, comprisinga microbial organism having a hexamethylenediamine (HMDA) pathwaycomprising at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA, said HMDApathway comprising a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase,a 3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a3-oxo-1-carboxyheptanal aminating oxidoreductase, a3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase, a 3-oxopimelate kinase, a5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming),a 3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelateligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ahomolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase(aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate aminating oxidoreductase.
 147. The non-naturallyoccurring microbial organism of claim 146, wherein said HMDA pathwaycomprises a pathway selected from the group consisting of: (i) aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ora homolysine decarboxylase; (ii) a glutaryl-CoA beta-ketothiolase, a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate kinase, a5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal7-aminotransferase, a 3-oxo-1-carboxyheptanal 7-aminatingoxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase, a3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase;(iii) a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a5-oxopimeloyl-CoA reductase (aldehyde forming), a3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate3-aminotransferase, a 3-oxo-7-aminoheptanoate 3-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysinedecarboxylase; (iv) comprises a glutaryl-CoA beta-ketothiolase, a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate reductase, a3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or homolysinedecarboxylase; (v) a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoAhydrolase, a 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate7-aminotransferase, a 3-amino-7-oxoheptanoate 7-aminatingoxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysinedecarboxylase; (vi) a glutaryl-CoA beta-ketothiolase, a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate CoA transferase or a3-oxopimelate CoA ligase, a 5-oxopimeloyl-CoA reductase (aldehydeforming), a 3-oxo-1-carboxyheptanal 3-aminotransferase, a3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, ora homolysine decarboxylase; (vii) a glutaryl-CoA beta-ketothiolase, a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase, a 3-aminopimelate reductase, a3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase,or a homolysine decarboxylase; (viii) a glutaryl-CoA beta-ketothiolase,a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysinedecarboxylase; (ix) a glutaryl-CoA beta-ketothiolase, a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a3-oxopimelate aminating oxidoreductase, a 3-aminopimelate CoAtransferase, a 3-aminopimelate CoA ligase, a 5-aminopimeloyl-CoAreductase (aldehyde forming), a 3-amino-7-oxoheptanoate 2,3-aminomutase,a 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase; (x) aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase,a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase;(xi) a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate CoA transferase, a 3-aminopimelate CoAligase, a 5-aminopimeloyl-CoA reductase (aldehyde forming), a3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoateaminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or ahomolysine decarboxylase; (xii) a glutaryl-CoA beta-ketothiolase, a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase, a3-oxopimeloyl-CoA ligase, a 3-oxopimelate aminotransferase, a3-oxopimelate aminating oxidoreductase, a 3-aminopimelate kinase, a5-aminopimeloylphosphonate reductase, a 3-amino-7-oxoheptanoate7-aminotransferase, a 3-amino-7-oxoheptanoate aminating oxidoreductase,a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase;(xiii) a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase,a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase; (xiv) aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelatekinase, a 6-aminopimeloylphosphonate reductase, a2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoateaminating oxidoreductase, or a homolysine decarboxylase; and (xv) aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, a 3-oxopimelate aminatingoxidoreductase, a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoAtransferase, 2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase(aldehyde forming), a 2-amino-7-oxoheptanoate 7-aminotransferase,2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysinedecarboxylase. 148-161. (canceled)
 162. The non-naturally occurringmicrobial organism of claim 146, wherein said microbial organismcomprises a set of exogenous nucleic acids encoding HMDA pathwayenzymes, said set of exogenous nucleic acids encoding: (i) aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 7-aminotransferase ora 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase; (ii) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminatingoxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;(iii) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a5-oxopimeloyl-CoA reductase (aldehyde forming); a3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferaseor a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;(iv) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase ora 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; anda homolysine decarboxylase; (v) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminatingoxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;(vi) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a5-oxopimeloyl-CoA reductase (aldehyde forming); a3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferaseor 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;(vii) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase; (viii) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase; (ix) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoAtransferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoAreductase (aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase;a 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase; (x) aglutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminatingoxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and ahomolysine decarboxylase; (xi) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoAtransferase or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoAreductase (aldehyde forming); a 3-amino-7-oxoheptanoate7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase;a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;(xii) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or a 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonatereductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a3-amino-7-oxoheptanoate aminating oxidoreductase; a3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase;(xiii) a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase,a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelatereductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase; (xiv) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate2,3-aminomutase; a 2-aminopimelate kinase; a 6-aminopimeloylphosphonatereductase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysinedecarboxylase; or (xv) a glutaryl-CoA beta-ketothiolase; a3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a3-oxopimelate aminating oxidoreductase; a 3-aminopimelate2,3-aminomutase; a 2-aminopimelate CoA transferase or 2-aminopimelateCoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde forming); a2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoateaminating oxidoreductase; and a homolysine decarboxylase. 163-178.(canceled)
 179. A non-naturally occurring microbial organism, comprisinga microbial organism having a hexamethylenediamine (HMDA) pathwaycomprising at least one exogenous nucleic acid encoding a HMDA pathwayenzyme expressed in a sufficient amount to produce HMDA, said HMDApathway comprising: (i) a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoateaminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminatingoxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoatedecarboxylase, a 6-aminohexanal aminotransferase or a 6-aminohexanalaminating oxidoreductase; or (ii) a 2-amino-7-oxosubarate keto-aciddecarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a 6-aminohexanalaminating oxidoreductase, a 6-aminohexanal aminotransferase, a2-amino-7-oxoheptanoate aminating oxidoreductase, a2-amino-7-oxoheptanoate aminotransferase, a homolysine decarboxylase, a2-amino-7-oxosubarate amino acid decarboxylase, a2-oxo-7-aminoheptanoate aminating oxidoreductase, a2-oxo-7-aminoheptanoate aminotransferase, a 2-oxo-7-aminoheptanoatedecarboxylase, a 2-amino-7-oxosubarate aminating oxidoreductase, a2-amino-7-oxosubarate aminotransferase or a 2,7-diaminosubaratedecarboxylase. 180-184. (canceled)
 185. The non-naturally occurringmicrobial organism of claim 179, wherein said microbial organismcomprises a set of exogenous nucleic acids encoding HMDA pathway enzymesexpressed in a sufficient amount to produce HMDA, said set of exogenousnucleic acids encoding: (i) a 2-oxo-4-hydroxy-7-aminoheptanoatealdolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoateaminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;and a homolysine decarboxylase; (ii) a 2-oxo-4-hydroxy-7-aminoheptanoatealdolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoatedecarboxylase; and a 6-aminohexanal aminotransferase or 6-aminohexanalaminating oxidoreductase; (iii) a 2-amino-7-oxosubarate aminatingoxidoreductase or 2-amino-7-oxosubarate aminotransferase; a2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase; (iv)a 2-amino-7-oxosubarate amino acid decarboxylase; a2-oxo-7-aminoheptanoate aminating oxidoreductase or a2-oxo-7-aminoheptanoate aminotransferase; and a homolysinedecarboxylase; (v) a 2-amino-7-oxosubarate amino acid decarboxylase; a2-oxo-7-aminoheptanoate decarboxylase; and a 6-aminohexanal aminatingoxidoreductase or a 6-aminohexanal aminotransferase; (vi) a2-amino-7-oxosubarate keto-acid decarboxylase; a 2-amino-7-oxoheptanoatedecarboxylase; and a 6-aminohexanal aminating oxidoreductase or a6-aminohexanal aminotransferase; or (vii) a 2-amino-7-oxosubarateketo-acid decarboxylase; a 2-amino-7-oxoheptanoate aminatingoxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and ahomolysine decarboxylase. 186-205. (canceled)
 206. The non-naturallyoccurring microbial organism of claim 179, part (ii), further comprisinga 2-amino-7-oxosubarate pathway comprising at least one exogenousnucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme expressedin a sufficient amount to produce 2-amino-7-oxosubarate, said2-amino-7-oxosubarate pathway comprising a2-amino-5-hydroxy-7-oxosubarate aldolase, a2-amino-5-hydroxy-7-oxosubarate dehydratase, or a2-amino-5-ene-7-oxosubarate reductase. 207-209. (canceled)
 210. Thenon-naturally occurring microbial organism of claim 185, part (iii)further comprising a 2-amino-7-oxosubarate pathway comprising a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, said second set of exogenous nucleic acidsencoding a 2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase. 211-213. (canceled)
 214. Thenon-naturally occurring microbial organism of claim 185, part (iv),further comprising a 2-amino-7-oxosubarate pathway comprising a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, said second set of exogenous nucleic acidsencoding a 2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase. 215-217. (canceled)
 218. Thenon-naturally occurring microbial organism of claim 185, part (v),further comprising a 2-amino-7-oxosubarate pathway comprising a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, said second set of exogenous nucleic acidsencoding a 2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase. 219-221. (canceled)
 222. Thenon-naturally occurring microbial organism of claim 185, part (vi),further comprising a 2-amino-7-oxosubarate pathway comprising a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, said second set of exogenous nucleic acidsencoding a 2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase. 223-225. (canceled)
 226. Thenon-naturally occurring microbial organism of claim 185, part (vii),further comprising a 2-amino-7-oxosubarate pathway comprising a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, said second set of exogenous nucleic acidsencoding a 2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase. 227-228. (canceled)
 229. Anon-naturally occurring microbial organism, comprising a microbialorganism having a 6-aminocaproic acid (6-ACA) pathway comprising atleast one exogenous nucleic acid encoding a 6-ACA pathway enzymeexpressed in a sufficient amount to produce 6-ACA, said 6-ACA pathwaycomprising a pathway selected from the group consisting of: (i) aglutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an3-oxo-6-aminopimeloyl-CoA oxidoreductase, a3-hydroxy-6-aminopimeloyl-CoA dehydratase, a6-amino-7-carboxyhept-2-enoyl-CoA reductase, a 6-aminopimeloyl-CoAreductase (aldehyde forming), or a 2-aminopimelate decarboxylase; (ii) aglutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase,a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase;(iii) a homolysine 2-monooxygenase; and (iv) a 2-amino-7-oxosubarateketo-acid decarboxylase, a 2-amino-7-oxoheptanoate decarboxylase, a2-amino-7-oxoheptanoate oxidoreductase, a 2-aminopimelate decarboxylase,a 6-aminohexanal oxidoreductase, a 2-amino-7-oxoheptanoatedecarboxylase, or a 2-amino-7-oxosubarate amino acid decarboxylase.230-234. (canceled)
 235. The non-naturally occurring microbial organismof claim 229, wherein said microbial organism comprises a set ofexogenous nucleic acids encoding 6-ACA pathway enzymes expressed in asufficient amount to produce 6-ACA, said set of exogenous nucleic acidsencoding: (i) a glutamyl-CoA transferase or a glutamyl-CoA ligase; abeta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase; a3-hydroxy-6-aminopimeloyl-CoA dehydratase; a6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoAreductase (aldehyde forming); and a 2-aminopimelate decarboxylase; (ii)a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a3-oxopimelate aminotransferase or 3-oxopimelate aminatingoxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelatedecarboxylase; (iii) a 2-amino-7-oxosubarate keto-acid decarboxylase; a2-amino-7-oxoheptanoate oxidoreductase; and a 2-aminopimelatedecarboxylase; (iv) a 2-amino-7-oxosubarate keto-acid decarboxylase; a2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanaloxidoreductase; or (v) a 2-amino-7-oxosubarate amino acid decarboxylase;a 2-amino-7-oxoheptanoate decarboxylase; and a 6-aminohexanaloxidoreductase. 236-257. (canceled)
 258. The non-naturally occurringmicrobial organism of claim 229, part (iv), further comprising a2-amino-7-oxosubarate pathway comprising at least one exogenous nucleicacid encoding a 2-amino-7-oxosubarate pathway enzyme expressed in asufficient amount to produce 2-amino-7-oxosubarate, said2-amino-7-oxosubarate pathway comprising a2-amino-5-hydroxy-7-oxosubarate aldolase, a2-amino-5-hydroxy-7-oxosubarate dehydratase, or a2-amino-5-ene-7-oxosubarate reductase. 259-267. (canceled)
 268. Thenon-naturally occurring microbial organism of claim 235, part (v),further comprising a 2-amino-7-oxosubarate pathway comprising a secondset of exogenous nucleic acids encoding 2-amino-7-oxosubarate pathwayenzymes expressed in a sufficient amount to produce2-amino-7-oxosubarate, said second set of exogenous nucleic acidsencoding a 2-amino-5-hydroxy-7-oxosubarate aldolase; a2-amino-5-hydroxy-7-oxosubarate dehydratase; and a2-amino-5-ene-7-oxosubarate reductase. 269-282. (canceled)
 283. Thenon-naturally occurring microbial organism of any one of claims 135, 146and 179, wherein said HDMA pathway comprises at least two, three or fourexogenous nucleic acids.
 284. The non-naturally occurring microbialorganism of claim 95 or 229, wherein said 6-ACA pathway comprises atleast two, three or four exogenous nucleic acids.
 285. The non-naturallyoccurring microbial organism of any one of claims 95, 135, 146, 179 and229, wherein said exogenous nucleic acid is a heterologous nucleic acid.286. The non-naturally occurring microbial organism of any one of claims95, 135, 146, 179 and 229, wherein said non-naturally occurringmicrobial organism is in a substantially anaerobic culture medium. 287.A method for producing 6-aminocaproic acid (6-ACA), comprising culturinga non-naturally occurring microbial organism of claim 95 or 229 underconditions and for a sufficient period of time to produce 6-ACA, whereinsaid conditions comprise substantially anaerobic culture conditions.288. A method for producing hexamethylenediamine (HMDA), comprisingculturing a non-naturally occurring microbial organism of any one ofclaims 135, 146, and 179 under conditions and for a sufficient period oftime to produce HMDA, wherein said conditions comprise substantiallyanaerobic culture conditions.